Fan Yuying, Ren Xiaoli, Wang Yingxue, Xu Enshuang, Wang Shuang, Ge Ruidong, Liu Yun
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.
Oncol Lett. 2021 Dec;22(6):852. doi: 10.3892/ol.2021.13113. Epub 2021 Oct 26.
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 G/G 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.
作为一种抗糖尿病药物，二甲双胍已被证明具有抗肿瘤作用。然而，其降低肿瘤形成（包括犬乳腺肿瘤，CMGT）的相关机制尚未完全阐明。本研究的目的是评估二甲双胍诱导CMGT细胞凋亡和细胞周期停滞的能力，并确定这些作用背后的途径。用二甲双胍处理后，通过细胞计数试剂盒-8分析评估细胞活力。随后，通过流式细胞术评估凋亡和细胞周期进程，并检测相关蛋白的表达。然后使用蛋白质印迹分析研究经典的AMP激活蛋白激酶（AMPK）、蛋白激酶B（AKT）、雷帕霉素靶蛋白（mTOR）和真核翻译起始因子4E结合蛋白1（4E-BP1）的表达水平。二甲双胍以浓度依赖的方式抑制CHMm细胞的增殖。具体而言，二甲双胍诱导细胞周期停滞在G/G期，伴随着p21和p27表达增加，细胞周期蛋白D1和细胞周期蛋白依赖性激酶4表达降低。在CHMm细胞中观察到明显的凋亡水平，同时伴有caspase-3激活和聚（ADP-核糖）聚合酶的裂解。此外，Bcl-2水平降低，Bax水平升高。相关信号分子的表达表明，二甲双胍显著增加CHMm细胞中AMPK的磷酸化，并降低磷酸化（p-）AKT、p-mTOR和p-4E-BP1的水平，而化合物C可逆转这些变化。这些发现表明，二甲双胍可能是一种治疗CMGT的潜在药物，通过AMPK/AKT/mTOR信号通路发挥作用。
