title: Metformin can block precancerous progression to invasive tumors of bladder through inhibiting STAT3-mediated signaling pathways.
abstract: BACKGROUND
  Metformin is the first line of oral antidiabetic drug in the biguanide class for treatment of type 2 diabetes. Increasing evidence has suggested that it is a potential anti-tumor drug. However, the mechanisms underlying inhibiting tumor development remain elusive, especially in bladder tumors.
  
  
  METHODS
  T24 and J82 cell lines were used as an in vitro model, and 24 female SD rats were used to build an N-methyl-N-nitrosourea (MNU)-induced orthotopic rat bladder cancer model. Transfection of lentivirus-based shRNA was used to construct the STAT3-KNOCKDOWN T24 cell line. After metformin treatment, the viability of bladde cancer cells was determined by CCK8. Cell cycle distribution and apoptosis were assessed by flow cytometry. The migration and invasion abilities of cells were evaluated by wound healing and transwell asssays. The inactivation of stat3 pahtway was examined by qRTPCR, western blot and Immunofluorescence.
  
  
  RESULTS
  Metformin can effectively inhibit precancerous progression to invasive cancer in an MNU-induced rat orthotopic bladder tumor model, although it could not completely suppress normal cells transforming into tumor cells. While the MNU could induce 50 % rats (4/8) to develop invasive bladder cancers, the rats co-administrated with metformin failed to develop invasive tumors but retained at precancerous or non-invasive stages, exhibiting as dysplasia, papillary tumor and/or carcinoma in situ (CIS). Accordingly, phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is a well known oncogene, was significantly inhibited in the tumors of rats treated with metformin. In vitro experiments revealed that the metformin could efficiently inhibit STAT3 activation, which was associated with the cell cycle arrest, reduction of cell proliferation, migration and invasiveness, and increase in apoptotic cell death of bladder cancer cell lines.
  
  
  CONCLUSIONS
  These findings provide for the first time the evidence that metformin can block precancerous lesions progressing to invasive tumors through inhibiting the activation of STAT3 pathway, and may be used for treatment of the non-invasive bladder cancers to prevent them from progression to invasive tumors.
authors.affiliation: Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. panqi0114@163.com.
authors.forename: Qi
authors.initials: Q
authors.lastname: Pan
authors.affiliation: Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. ygl0511@126.com.
authors.forename: Guo-Liang
authors.initials: GL
authors.lastname: Yang
authors.affiliation: Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. 446598465@qq.com.
authors.forename: Jiang-Hua
authors.initials: JH
authors.lastname: Yang
authors.affiliation: Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. lin_shilong@126.com.
authors.forename: Shi-Long
authors.initials: SL
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Oncogene and Related Genes, Shanghai, China. hiliuning@163.com.
authors.forename: Ning
authors.initials: N
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Oncogene and Related Genes, Shanghai, China. 1042846092@qq.com.
authors.forename: Shan-Shan
authors.initials: SS
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Oncogene and Related Genes, Shanghai, China. happyliumy@163.com.
authors.forename: Meng-Yao
authors.initials: MY
authors.lastname: Liu
authors.affiliation: Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. 13061759248@163.com.
authors.forename: Lian-Hua
authors.initials: LH
authors.lastname: Zhang
authors.affiliation: Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. hyrrenji2@yahoo.com.cn.
authors.forename: Yi-Ran
authors.initials: YR
authors.lastname: Huang
authors.affiliation: Department of Pathology, Ohio State University School of Medicine, Columbus, OH, USA. rulongshen@gmail.comc.
authors.forename: Ru-long
authors.initials: RL
authors.lastname: Shen
authors.affiliation: Department of Pathology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. liuqiang@163.com.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Oncogene and Related Genes, Shanghai, China. jianxingao@sjtu.edu.cn.|Laboratory of Tumorigenesis and Immunity, Clinical Stem Cell Research Center, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. jianxingao@sjtu.edu.cn.
authors.forename: Jian-Xin
authors.initials: JX
authors.lastname: Gao
authors.affiliation: Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. bojuanjie@yeah.net.
authors.forename: Juan-Jie
authors.initials: JJ
authors.lastname: Bo
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D018450:Disease Progression; D004354:Drug Screening Assays, Antitumor; D005260:Female; D006801:Humans; D008687:Metformin / Q000494:pharmacology*; D009361:Neoplasm Invasiveness; D011230:Precancerous Conditions / Q000188:drug therapy* / Q000473:pathology; D017207:Rats, Sprague-Dawley; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000473:pathology
chemical_list: D000970:Antineoplastic Agents; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D008687:Metformin