Metformin inhibits the proliferation of canine mammary gland tumor cells through the AMPK/AKT/mTOR signaling pathway <em>in vitro</em>

Fan,Yuying; Ren,Xiaoli; Wang,Yingxue; Xu,Enshuang; Wang,Shuang; Ge,Ruidong; Liu,Yun

doi:10.3892/ol.2021.13113

Metformin inhibits the proliferation of canine mammary gland tumor cells through the AMPK/AKT/mTOR signaling pathway in vitro

Authors:
- Yuying Fan
- Xiaoli Ren
- Yingxue Wang
- Enshuang Xu
- Shuang Wang
- Ruidong Ge
- Yun Liu
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Affiliations: Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150000, P.R. China, College of Veterinary Medicine, Henan University of Animal Husbandry and Economy, Zhengzhou, Henan 450046, P.R. China, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163000, P.R. China
Published online on: October 26, 2021 https://doi.org/10.3892/ol.2021.13113
Article Number: 852
Copyright: © Fan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

As an anti‑diabetic drug, metformin has been demonstrated to exhibit antitumor effects. However, the mechanisms involved in decreasing tumor formation, including canine mammary gland tumors (CMGTs), are not well elucidated. The aim of the present study was to evaluate the ability of metformin to induce apoptosis and cell cycle arrest in CMGT cells, as well as identifying the pathways underlying these effects. Cell viability was assessed by Cell Counting Kit‑8 analysis following treating with metformin. Subsequently, apoptosis and cell cycle progression were assessed by flow cytometry, and the expression of associated proteins was examined. Expression levels of classical AMP‑activated protein kinase (AMPK), protein kinase B (AKT), mechanistic target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E‑binding protein 1 (4E‑BP1) were then investigated using western blot analysis. Metformin inhibited the proliferation of CHMm cells in a concentration‑dependent manner. Specifically, metformin induced cell cycle arrest in the G₀/G₁ phases, accompanied by increased expression of p21 and p27, and decreased expression of cyclin D1 and cyclin‑dependent kinase 4. Marked levels of apoptosis were observed in CHMm cells alongside the activation of caspase‑3 and cleavage of poly(ADP‑ribose) polymerase. Also, the level of Bcl‑2 was decreased, and that of Bax was increased. The expression of associated signaling molecules revealed that metformin markedly increased the phosphorylation of AMPK in CHMm cells, and decreased the levels of phosphorylated (p‑)AKT, p‑mTOR and p‑4E‑BP1, while Compound C reversed these changes. These findings demonstrated that metformin may be a potential therapeutic agent for CMGTs, acting via the AMPK/AKT/mTOR signaling pathway.

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