Background: Canine mammary tumors are one of the most common tumors in female dogs, and the development of effective therapeutic agents is a critical clinical challenge. Chrysin, a natural flavonoid compound, has been reported to exhibit anticancer activity in various human cancers through the regulation of cell proliferation and apoptosis. However, the anticancer effects of chrysin on canine mammary tumors have not yet been fully elucidated. Therefore, this study investigated the antiproliferative and apoptosis-inducing effects of chrysin on CMT-U27 cells derived from canine mammary tumors. Methods: Antiproliferative activity was confirmed by treating CMT-U27 cells with chrysin at concentrations of 0-10 μM and evaluating cell viability. Cell proliferation capacity was assessed using Ki-67 immunocytochemistry. The expression levels of cell cycle-related proteins, including CDK2, CDK4, cyclin D, and cyclin E, were analyzed by Western blotting. Additionally, apoptosis-related proteins, including phosphorylated p53 and BCL2, were evaluated to investigate the apoptotic activity of chrysin treatment. Results: Chrysin treatment significantly reduced the viability and migration of CMT-U27 cells and decreased the number of Ki-67-positive cells in a dose-dependent manner. Following chrysin treatment, the expression of CDK2, CDK4, cyclin D, and cyclin E was significantly reduced, indicating that cell cycle progression was inhibited. Additionally, chrysin increased the expression of phosphorylated p53 and decreased BCL2 expression. Conclusions: Chrysin exhibited significant anticancer effects in canine mammary tumor-derived CMT-U27 cells through the inhibition of cell proliferation, accompanied by G2/M phase accumulation and downregulation of cyclin-CDK complex components. In addition, chrysin altered apoptosis-related signaling molecules, including p53 and BCL2. These results suggest that chrysin may have potential as a therapeutic candidate for canine mammary tumors.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Cell culture and chrysin treatment
    Cell viability assay
    Immunostaining
    Flow cytometry
    Cell-migration assay
    Western blotting
    Statistical analysis
RESULTS
    Chrysin suppresses cell viability and proliferation inCMT-U27 cells
    Chrysin induces cell cycle arrest in CMT-U27 cells
    Chrysin induces apoptosis and inhibits migration inCMT-U27 cells
DISCUSSION
CONCLUSION
REFERENCES

• Hyeon Woo Sim(Department of Animal Biotechnology, College of Life Science, Sangji University, Wonju 26339, Korea)
• Kwanghyeon Cho(Department of Livestock, Korea National University of Agriculture and Fisheries, Jeonju 54874, Korea)
• Ran Lee(Department of Livestock, Korea National University of Agriculture and Fisheries, Jeonju 54874, Korea)
• Won-Young Lee(Department of Livestock, Korea National University of Agriculture and Fisheries, Jeonju 54874, Korea) Corresponding author
• Jin-Yeong Kim(Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chungju 27478, Korea)
• Hyun-Jung Park(Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chungju 27478, Korea) Corresponding author
• Eun-Jung Kim(Department of Biomedical Laboratory Science, College of Health Sciences, Sangji University, Wonju 26339, Korea)