| Canine mammary gland tumors(CMGTs)are the most frequent tumors in female dogs,associated with age,intactness,fertility and genetic factor similar to human breast cancer and regarded as a perfect model to study human breast cancer for similar pathogenesis,grade and diagnosis,thus it was urgent for human and canine breast cancer to search for a safe and effective chemotherapeutics.Metformin(1,1-dimethylbiguanide)is widely applied to treat type 2 diabetes,and has become the first-line hypoglycemic drug since the 1950 s because of its high safety and great tolerability by the American Disease Association and the European Association for the Study of Diabetes.It has been previous indicated that metformin exhibits not just hypoglycemic effects,epidemiologic studies and meta-analysis have showed that metformin appeared more capable in reduction of related-cancer mortality and risk.The present study aims to investigate the possibility of using metformin’s to treat CMGTs by performing experiments on cell lines CHMm and CHMp,further to provide some basis for the research of breast cancer in dogs and human.Methods: CHMm and CHMp were derived from pleural effusion and primary mass of canine spontaneous mammary adenocarcinomas respectively,and analyzed for cell proliferation by CCK-8 assay and colony formation after treated with metformin.Then,induced apoptosis with fluorescein isothiocyanate(FITC)marker and cell cycle were measured with flow cytometry(FCM),related proteins were considered as further assessment.Eventually,classical AMPK,phoinositide 3-kinase(PI3K)expression level and of its potential downstream targets including Protein kinase B(PKB/Akt),and mammalian target of rapamycin(mTOR),were examined using western blot.Result: Metformin inhibited the proliferation and colony formation of CHMm and CHMp in a time-and concentration-dependent way,especially for CHMm from pleural effusion as metastatic tumor cells.Specifically,metformin induced cell cycle arrest in G0/G1 phases,accompanied by up-expression of p21,p27,and down-expression of cyclin D1 and cyclin-dependent kinase 4(CDK4).Remarkable apoptosis was observed in CHMm with the activation of caspase-3 and cleavage of PARP but not in CHMp.The expressions of related signal molecules results showed that metformin increased the phosphorylation expression of AMPK in CHMm dramatically rather than CHMp and inhibited activities of PI3K/Akt mTOR relevant proteins.Conclusion: Metformin-induced apoptosis and G0/G1 phase cell cycle arrest on CMGTs were mediated possibly by independent AMPK activation and inhibition of PI3K/Akt/m TOR.These findings show that metformin may be a potential therapeutic agent for CMGTs,which will providesome basis for the treatment of CMGTs and a perfect model for the research of human breast cancer. |
