title: Metformin affects the features of a human hepatocellular cell line (HepG2) by regulating macrophage polarization in a co-culture microenviroment.
abstract: OBJECTIVE
  Accumulating evidence suggests an association between diabetes and cancer. Inflammation is a key event that underlies the pathological processes of the two diseases. Metformin displays anti-cancer effects, but the mechanism is not completely clear. This study investigated whether metformin regulated the microenvironment of macrophage polarization to affect the characteristics of HepG2 cells and the possible role of the Notch-signalling pathway.
  
  
  METHODS
  RAW264.7 macrophages were cultured alone or co-cultured with HepG2 cells and treated with metformin. We analysed classical (M1) and alternative (M2) gene expression in RAW264.7 cells using quantitative real-time polymerase chain reaction. Changes in mRNA and protein expressions of Notch signalling in both cell types were also detected using quantitative real-time polymerase chain reaction and Western-blotting analyses. The proliferation, apoptosis and migration of HepG2 cells were detected using Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega Corporation, Fitchburg, WI, USA), Annexin V-FITC/PI (7SeaPharmTech, Shanghai, China) and the cell scratch assay, respectively.
  
  
  RESULTS
  Metformin induced single-cultured RAW264.7 macrophages with an M2 phenotype but attenuated the M2 macrophage differentiation and inhibited monocyte chemoattractant protein-1 (MCP-1) secretion in a co-culture system. The co-cultured group of metformin pretreatment activated Notch signalling in macrophages but repressed it inHepG2 cells. Co-culture also promoted the proliferation and migration of HepG2 cells. However, along with the enhanced apoptosis, the proliferation and the migration of HepG2 cells were remarkably inhibited in another co-culture system with metformin pretreatment.
  
  
  CONCLUSIONS
  Metformin can skew RAW264.7 macrophages toward different phenotypes according to changes in the microenvironment, which may affect the inflammatory conditions mediated by macrophages, induce apoptosis and inhibit the proliferation and migration of HepG2 cells. Notch signalling pathway is a potentially important mechanism in the regulation of metformin on macrophage polarization and the subsequent change of hepatoma cells. Copyright © 2015 John Wiley & Sons, Ltd.
authors.affiliation: Institute of Metabolism and Endocrinology, the Second Xiangya Hospital of Central South University, Changsha, Hunan, China.|Diabetes Center of Central South University, Changsha, Hunan, China.|National Clinical Research Center for Metabolic Diseases, Changsha, Hunan, China.
authors.forename: Miaojiao
authors.initials: M
authors.lastname: Chen
authors.affiliation: Institute of Metabolism and Endocrinology, the Second Xiangya Hospital of Central South University, Changsha, Hunan, China.|Metabolic Syndrome Research Center, the Second Xiangya Hospital of Central South University, Changsha, Hunan, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Metabolic Syndrome Research Center, the Second Xiangya Hospital of Central South University, Changsha, Hunan, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Hu
authors.affiliation: Institute of Metabolism and Endocrinology, the Second Xiangya Hospital of Central South University, Changsha, Hunan, China.|Diabetes Center of Central South University, Changsha, Hunan, China.|National Clinical Research Center for Metabolic Diseases, Changsha, Hunan, China.
authors.forename: Shiping
authors.initials: S
authors.lastname: Liu
authors.affiliation: Institute of Metabolism and Endocrinology, the Second Xiangya Hospital of Central South University, Changsha, Hunan, China.|Diabetes Center of Central South University, Changsha, Hunan, China.|National Clinical Research Center for Metabolic Diseases, Changsha, Hunan, China.
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects*; D018932:Chemokine CCL2 / Q000378:metabolism; D018920:Coculture Techniques; D056945:Hep G2 Cells; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008687:Metformin / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D007004:Hypoglycemic Agents; D008687:Metformin
keywords: Notch; hepatoma; inflammation; macrophage; metformin; polarization