Natural Product Epipyrone A from Epicoccum nigrum Exhibits Antiproliferative Activity on Canine Mammary Tumor Cells Through PI3K/Akt/mTOR Pathway Modulation

Canine mammary tumors (CMTs) are among the most frequent neoplasms in female dogs, with current therapeutic options being limited and non-standardized, prompting the search for alternative treatments such as fungal secondary metabolites. In this study, the fungal pigment Epipyrone A (Epy A) was first isolated from <i>Epicoccum nigrum</i> and then tested in vitro on two CMT cell lines, P114 and CF33. The compound significantly reduced cell viability in both lines in a concentration- and time-dependent manner (<i>p</i> < 0.05), with the strongest effect observed at 175 µg/mL after 48 h (<i>p</i> < 0.0001), while showing no cytotoxicity in MDCK non-tumor cells. Epy A also inhibited cell migration and increased total antioxidant capacity in P114 cells, accompanied by a significant reduction in ROS levels. Western blot analysis revealed modulation of the PI3K/Akt/mTOR pathway, crucial in CMT biology. Specifically, P114 cells showed downregulation of mTOR and p-Akt, indicating inhibition of proliferative signaling, whereas CF33 cells exhibited increased Akt and p-Akt alongside reduced mTOR, consistent with a compensatory feedback mechanism, probably linked to the changing in oxidative balance after treatment. Overall, these results identified Epy A as a promising natural molecule with potential applications in innovative therapeutic approaches for veterinary and comparative oncology.