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 Epicoccum nigrum 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 (p < 0.05), with the strongest effect observed at 175 µg/mL after 48 h (p < 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.