title: Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells.
abstract: Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration-dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.
authors.affiliation: Pharmacology Department, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil; Department of Physiological Sciences, State University of Londrina, Londrina, Paraná, Brazil.
authors.forename: Eveline A I F
authors.initials: EA
authors.lastname: Queiroz
authors.affiliation: Biology Department, Lakehead University, Thunder Bay, Ontario, Canada.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Puukila
authors.affiliation: Pharmacology Department, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Rosangela
authors.initials: R
authors.lastname: Eichler
authors.affiliation: Pharmacology Department, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Sandra C
authors.initials: SC
authors.lastname: Sampaio
authors.affiliation: Northern Ontario School of Medicine, Lakehead University, Thunder Bay, Ontario, Canada.
authors.forename: Heidi L
authors.initials: HL
authors.lastname: Forsyth
authors.affiliation: Northern Ontario School of Medicine, Lakehead University, Thunder Bay, Ontario, Canada; Biology Department, Lakehead University, Thunder Bay, Ontario, Canada.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Lees
authors.affiliation: Biorefining Research Institute, Lakehead University, Thunder Bay, Ontario, Canada.
authors.forename: Aneli M
authors.initials: AM
authors.lastname: Barbosa
authors.affiliation: Biorefining Research Institute, Lakehead University, Thunder Bay, Ontario, Canada.
authors.forename: Robert F H
authors.initials: RF
authors.lastname: Dekker
authors.affiliation: Pharmacology Department, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Zuleica B
authors.initials: ZB
authors.lastname: Fortes
authors.affiliation: Northern Ontario School of Medicine, Lakehead University, Thunder Bay, Ontario, Canada; Biology Department, Lakehead University, Thunder Bay, Ontario, Canada.
authors.forename: Neelam
authors.initials: N
authors.lastname: Khaper
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D006801:Humans; D006859:Hydrogen / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D008687:Metformin / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D016192:Resting Phase, Cell Cycle / Q000187:drug effects*
chemical_list: C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D007004:Hypoglycemic Agents; D009363:Neoplasm Proteins; D016877:Oxidants; D006859:Hydrogen; D008687:Metformin; D055372:AMP-Activated Protein Kinases