title: Anti-tumour effect of metformin in canine mammary gland tumour cells.
abstract: Metformin is an oral hypoglycaemic drug used in type 2 diabetes. Its pharmacological activity reportedly involves mitochondrial respiratory complex I, and mitochondrial respiratory complex inhibitors have a strong inhibitory effect on the growth of metastatic canine mammary gland tumour (CMGT) cell lines. It is hypothesised that metformin has selective anti-tumour effects on metastatic CMGT cells. The aim of this study was to investigate the in vitro effect of metformin on cell growth, production of ATP and reactive oxygen species (ROS), and the AMP-activated protein kinase (AMPK) mammalian target of rapamycin (mTOR) pathway in two CMGT clonal cell lines with different metastatic potential. In addition, transcriptome analysis was used to determine cellular processes disrupted by metformin and in vivo anti-tumour effects were examined in a mouse xenograft model. Metformin inhibited CMGT cell growth in vitro, with the metastatic clone (CHMp-5b) displaying greater sensitivity. ATP depletion and ROS elevation were observed to a similar extent in the metastatic and non-metastatic (CHMp-13a) cell lines after metformin exposure. However, subsequent AMPK activation and mTOR pathway inhibition were prominent only in metformin-insensitive non-metastatic cells. Microarray analysis revealed inhibition of cell cycle progression by metformin treatment in CHMp-5b cells, which was further confirmed by Western blotting and cell cycle analysis. Additionally, metformin significantly suppressed tumour growth in xenografted metastatic CMGT cells. In conclusion, metformin exhibited an anti-tumour effect in metastatic CMGT cells through AMPK-independent cell cycle arrest. Its mechanism of action differed in the non-metastatic clone, where AMPK activation and mTOR inhibition were observed.
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Saeki
authors.affiliation: Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Tsuboi
authors.affiliation: Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Sugano
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Yoshitake
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: S M
authors.initials: SM
authors.lastname: Ong
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Saito
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Matsumoto
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Fujita
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Nishimura
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: anakaga@mail.ecc.u-tokyo.ac.jp.
authors.forename: T
authors.initials: T
authors.lastname: Nakagawa
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004283:Dog Diseases / Q000188:drug therapy*; D004285:Dogs; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007381:Intermediate Filament Proteins; D015674:Mammary Neoplasms, Animal / Q000188:drug therapy*; D008687:Metformin / Q000494:pharmacology*; D051379:Mice; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000188:drug therapy; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D007004:Hypoglycemic Agents; D007381:Intermediate Filament Proteins; C452701:RANDAM-1 protein, mouse; D017382:Reactive Oxygen Species; D008687:Metformin; D055372:AMP-Activated Protein Kinases
keywords: AMP-activated protein kinase (AMPK); Canine; Mammary gland tumour; Metastasis; Metformin; Mitochondria