Synergistic Antitumor Effects of Ivermectin and Metformin in Canine Breast Cancer via PI3K/AKT/mTOR Pathway Inhibition

Ivermectin (IVM) is a macrolide antiparasitic drug, and Metformin (MET) is a biguanide oral hypoglycemic drug. Studies have shown that both of them have obvious anti-tumor effects, but there have been no reports on the combined treatment of Canine breast tumors.This report aimed to investigate the effectiveness and the possible mechanism of drug combination on Canine breast cancers. Mouse breast tumor cells (4T1) and canine breast tumor cell (CMT-1211) were respec-tively treated with IVM, MET and their combination, and then cell viability was assessed. After that transcriptomic analysis was performed to study the action pathway of the drug combination on anti-tumor. Reactive oxygen species (ROS) levels were detected by flow cytometry, and au-tophagosome formation was observed by transmission electron microscopy (TEM). Immunoflu-orescence detected the cytoplasmic translocation of LC3B and P62 into the nucleus. Western blot detected the protein expressions of LC3B, P62, Beclin1, Bcl-2, p-PI3K, p-AKT, and p-mTOR. Our transcriptomic analysis showed that the combination of IVM and MET regulated the expression of autophagy-related genes and pathways, including the PI3K/AKT/mTOR signalling pathway. Our in vitro experiments showed that the combination of two drugs had a considerably significant effect on cytotoxicity, ROS levels, and the formation of autophagosomes compared to each drug alone. Meanwhile, in vivo experiments showed that It was observed that IVM combined with MET had obvious inhibitory effect on tumor growth in canine breast tumor xenografts. This study concluded that IVM with MET activated autophagy, which killed breast cancer cells by inhibiting the activation of the PI3K/AKT/mTOR pathway and promoting the excessive accumulation of ROS. It offers a theoretical foundation for the synergistic effects of MET and IVM to suppress breast cancer cell activity.