Tumor Biology

, Volume 37, Issue 4, pp 5337–5346 | Cite as

Mechanism of metformin action in MCF-7 and MDA-MB-231 human breast cancer cells involves oxidative stress generation, DNA damage, and transforming growth factor β1 induction

  • Poliana Camila Marinello
  • Thamara Nishida Xavier da Silva
  • Carolina Panis
  • Amanda Fouto Neves
  • Kaliana Larissa Machado
  • Fernando Henrique Borges
  • Flávia Alessandra Guarnier
  • Sara Santos Bernardes
  • Júlio Cesar Madureira de-Freitas-Junior
  • José Andrés Morgado-Díaz
  • Rodrigo Cabral Luiz
  • Rubens Cecchini
  • Alessandra Lourenço Cecchini
Original Article


The participation of oxidative stress in the mechanism of metformin action in breast cancer remains unclear. We investigated the effects of clinical (6 and 30 μM) and experimental concentrations of metformin (1000 and 5000 μM) in MCF-7 and in MDA-MB-231 cells, verifying cytotoxicity, oxidative stress, DNA damage, and intracellular pathways related to cell growth and survival after 24 h of drug exposure. Clinical concentrations of metformin decreased metabolic activity of MCF-7 cells in the MTT assay, which showed increased oxidative stress and DNA damage, although cell death and impairment in the proliferative capacity were observed only at higher concentrations. The reduction in metabolic activity and proliferation in MDA-MB-231 cells was present only at experimental concentrations after 24 h of drug exposition. Oxidative stress and DNA damage were induced in this cell line at experimental concentrations. The drug decreased cytoplasmic extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT and increased nuclear p53 and cytoplasmic transforming growth factor β1 (TGF-β1) in both cell lines. These findings suggest that metformin reduces cell survival by increasing reactive oxygen species, which induce DNA damage and apoptosis. A relationship between the increase in TGF-β1 and p53 levels and the decrease in ERK1/2 and AKT was also observed. These findings suggest the mechanism of action of metformin in both breast cancer cell lineages, whereas cell line specific undergoes redox changes in the cells in which proliferation and survival signaling are modified. Taken together, these results highlight the potential clinical utility of metformin as an adjuvant during the treatment of luminal and triple-negative breast cancer.


Metformin MCF-7 MDA-MB-231 Oxidative stress Breast cancer 



Extracellular signal-regulated kinases 1 and 2


Protein kinase B


Transforming growth factor β1


Adenosine-5′-monophosphate-activated protein kinase


Mammalian target of rapamycin


Human epidermal growth factor receptor 2


Triple-negative breast cancer


2-(3,5-Diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole bromide


Phosphate-buffered saline


Ethidium bromide


Acridine orange






Analysis of variance


Superoxide dismutase


Oxidative stress


Reactive oxygen species



The authors are grateful to J.A. Vargas and P.S.R. Dionízio-Filho, from the Department of General Pathology of the State University of Londrina, for their excellent technical assistance.

Compliance with ethical standards

Conflicts of interest


Supplementary material

13277_2015_4395_Fig7_ESM.gif (341 kb)
Supplementary Figure 1

Metformin increases nuclear p53 levels and cytoplasmic TGF-β1 levels and reduces cytoplasmic ERK1/2 and AKT in human breast cancer cells. Immunocytochemistry analysis of MCF-7 cells (a) and MDA-MB-231 cells (b) exposed to different metformin concentrations (6, 30, 1000, and 5000 μM) for 24 h. Illustrative panel showing a picture selected for each experimental condition. For the metformin 5000 μM, where the effects were more pronounced, a picture focusing on a single cell was selected (GIF 340 kb).

13277_2015_4395_MOESM1_ESM.tif (3.9 mb)
High Resolution Image (TIF 3950 kb).


  1. 1.
    Aksoy S, Sendur MA, Altundag K. Demographic and clinico-pathological characteristics in patients with invasive breast cancer receiving metformin. Med Oncol. 2013;30(2):590–6.CrossRefPubMedGoogle Scholar
  2. 2.
    Dowling RJ, Niraula S, Stambolic V, Goodwin PJ. Metformin in cancer: translational challenges. J Mol Endocrinol. 2012;48(3):31–43.CrossRefGoogle Scholar
  3. 3.
    Dowling RJ, Zakikhani M, Fantus IG, Pollak M, Sonenberg N. Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res. 2007;67(22):10804–12.CrossRefPubMedGoogle Scholar
  4. 4.
    Martinez-Outschoorn UE, Goldberg A, Lin Z, Ko Y, Flomenberg N, Wang C, et al. Anti-estrogen resistance in breast cancer is induced by the tumor microenvironment and can be overcome by inhibiting mitochondrial function in epithelial cancer cells. Cancer Biol Ther. 2011;12(10):924–38.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Campagnoli C, Berrino F, Venturelli E, Abbà C, Biglia N, Brucato T, et al. Metformin decreases circulating androgen and estrogen levels in nondiabetic women with breast cancer. Clin Breast Cancer. 2013;13(6):433–8.CrossRefPubMedGoogle Scholar
  6. 6.
    Hadad SM, Hardie DG, Appleyard V, Thompson AM. Effects of metformin on breast cancer cell proliferation, the AMPK pathway and the cell cycle. Clin Transl Oncol. 2014;16(8):746–52.CrossRefPubMedGoogle Scholar
  7. 7.
    Ishibashi Y, Matsui T, Takeuchi M, Yamagishi S. Metformin inhibits advanced glycation end products (AGEs)-induced growth and VEGF expression in MCF-7 breast cancer cells by suppressing AGEs receptor expression via AMP-activated protein kinase. Horm Metab Res. 2013;45(5):387–90.PubMedGoogle Scholar
  8. 8.
    Song CW, Lee H, Dings RP, Williams B, Powers J, Santos TD, et al. Metformin kills and radiosensitizes cancer cells and preferentially kills cancer stem cells. Sci Rep. 2012;2:362.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Malki A, Youssef A. Antidiabetic drug metformin induces apoptosis in human MCF breast cancer via targeting ERK signaling. Oncol Res. 2011;19(6):275–85.CrossRefPubMedGoogle Scholar
  10. 10.
    Gago-Dominguez M, Jiang X, Castelao JE. Lipid peroxidation, oxidative stress genes and dietary factors in breast cancer protection: a hypothesis. Breast Cancer Res. 2007;9:201–12.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Panis C, Herrera AC, Victorino VJ, Campos FC, Freitas LF, De Rossi T, et al. Oxidative stress and hematological profiles of advanced breast cancer patients subjected to paclitaxel or doxorubicin chemotherapy. Breast Cancer Res Treat. 2012;133(1):89–97.CrossRefPubMedGoogle Scholar
  12. 12.
    Queiroz EAIF, Puukila S, Eichler R, Sampaio SC, Forsyth HL, Lees SJ, et al. Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells. Plos One. 2014;9(5):e98207.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ringnér M, Staaf J, Jonsson G. Nonfamilial breast cancer subtypes. Methods Mol Biol. 2013;973:279–95.CrossRefPubMedGoogle Scholar
  14. 14.
    Morris GJ, Naidu S, Topham AK, Guiles F, Xu Y, Mccue P, et al. Differences in breast carcinoma characteristics in newly diagnosed African-American and Caucasian patients: a single-institution compilation compared with the National Cancer Institute’s Surveillance, Epidemiology, and End Results database. Cancer. 2007;110(4):876–84.CrossRefPubMedGoogle Scholar
  15. 15.
    Barcellos-Hoff MH, Akhurst RJ. Transforming growth factor-β in breast cancer: too much, too late. Breast Cancer Res. 2009;11:202–8.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway in human cancer: rationale and promise. Cancer Cell. 2003;4(4):257–62.CrossRefPubMedGoogle Scholar
  17. 17.
    Miller LD, Smeds J, George J, Vega VB, Vergara L, Ploner A, et al. An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival. Proc Natl Acad Sci U S A. 2005;102(38):13550–5.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation cytotoxic assays. J Immunol Methods. 1983;65:55–63.CrossRefPubMedGoogle Scholar
  19. 19.
    Borenfreund E, Puerner JA. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J Tissue Cult Methods. 1984;9(1):7–9.CrossRefGoogle Scholar
  20. 20.
    Halliwell B, Gutteridge JMC. Free radical in biology and medicine. 4th ed. New York: Oxford University Press; 2007.Google Scholar
  21. 21.
    Michaeli A, Feitelson J. Reactivity of singlet oxygen toward amino acids and peptides. Photochem Photobiol. 1994;59:284–98.CrossRefPubMedGoogle Scholar
  22. 22.
    Simic MG. Peroxyl radical from oleic acid. In: Simic MG, editor. Autoxidation in food and biological systems. New York: Plenum; 1980. p. 17–26.CrossRefGoogle Scholar
  23. 23.
    Sun Y, Li Y, Wu H, Wu S, Wang YA, Luo D, et al. Effects of an indolocarbazole-derived CDK4 inhibitor on breast cancer cells. J Cancer. 2011;2:36–51.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–6.CrossRefPubMedGoogle Scholar
  25. 25.
    Hu ML. Measurement of protein thiol groups and glutathione in plasma. Methods Enzymol. 1994;233:380–5.CrossRefPubMedGoogle Scholar
  26. 26.
    Gonzalez-Flecha B, Llesuy S, Boveris A. Hydroperoxide-initiated chemiluminescence: an assay for oxidative stress in biopsies of heart, liver, and muscle. Free Radic Biol Med. 1991;10(2):93–100.CrossRefPubMedGoogle Scholar
  27. 27.
    Victorino VJ, Panis C, Campos FC, Cayres RC, Colado-Simão AN, Oliveira SR, et al. Decreased oxidant profile and increased antioxidant capacity in naturally postmenopausal women. AGE. 2013;35:1411–21.CrossRefPubMedGoogle Scholar
  28. 28.
    Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35:206–21.CrossRefPubMedGoogle Scholar
  29. 29.
    Hartmann A, Agurell E, Beevers S, Brendler-Schwaab S, Burlinson B, Clay P, et al. Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis. 2003;18(1):45–51.CrossRefPubMedGoogle Scholar
  30. 30.
    Wurth R, Barbieri F, Florio T. New molecules and old drugs as emerging approaches to selectively target human glioblastoma cancer stem cells. Biomed Res Int. 2014;2014:126586.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Lee H, Park HJ, Park CS, Oh ET, Choi BH, Williams B, et al. Response of breast cancer cells and cancer stem cells to metformin and hyperthermia alone or combined. PLoS One. 2014;9(2):e87979.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Lin YC, Wu MH, Wei TT, Lin YC, Huang WC, et al. Metformin sensitizes anticancer effect of dasatinib in head and neck squamous cell carcinoma cells through AMPK-dependent ER stress. Oncotarget. 2013;5(1):298–308.Google Scholar
  33. 33.
    Gonzalez-Ângulo AM, Meric-Bernstam F. Metformin: a therapeutic opportunity in breast cancer. Clin Cancer Res. 2012;16(6):1695–700.CrossRefGoogle Scholar
  34. 34.
    Zhuang Y, Miskimins WK. Metformin induces both caspase-dependent and poly(ADP-ribose) polymerase-dependent cell death in breast cancer cells. Mol Cancer Res. 2011;9(5):603–15.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Hadad SM, Hardie DG, Appleyard V, Thompson AM. Effects of metformin on breast cancer cell proliferation, the AMPK pathway and the cell cycle. Clin Transl Oncol. 2014;16:746–51.CrossRefPubMedGoogle Scholar
  36. 36.
    Zordoky BNM, Bark D, Soltys CL, Sung MM, Dyck JRB. The anti-proliferative effect of metformin in triple-negative MDA-MB-231 breast cancer cells is highly dependent on glucose concentration: implications for cancer therapy and prevention. Biochim Biophys Acta. 2014;1840(6):1943–57.CrossRefPubMedGoogle Scholar
  37. 37.
    Hasty P, Christy BA. p53 as an intervention target for cancer and aging. Pathobiol Aging Age Relat Dis. 2013;3:22702.CrossRefGoogle Scholar
  38. 38.
    Jin S, Levine AJ. The p53 functional circuit. J Cell Sci. 2001;114:4139–40.PubMedGoogle Scholar
  39. 39.
    Feng Z, Zhang H, Levine AJ, Jin S. The coordinate regulation of the p53 and mTOR pathways in cells. Proc Natl Acad Sci U S A. 2005;102(23):8204–9.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Santarpia L, Lippman SL, El-Naggar AK. Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy. Expert Opin Ther Targets. 2012;16(1):103–19. doi: 10.1517/14728222.2011.645805.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Krstic J, Trivanovic D, Mojsilovic S, Santibanez JF. Transforming growth factor-beta and oxidative stress interplay: implications in tumorigenesis and cancer progression. Oxid Med Cell Longev. 2015;2015(654594):15.Google Scholar
  42. 42.
    Qi S, den Hartog GJ, Bast A. Superoxide radicals increase transforming growth factor-beta1 and collagen release from human lung fibroblasts via cellular influx through chloride channels. Toxicol Appl Pharmacol. 2009;237(1):111–8.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Poliana Camila Marinello
    • 1
  • Thamara Nishida Xavier da Silva
    • 2
  • Carolina Panis
    • 3
  • Amanda Fouto Neves
    • 1
  • Kaliana Larissa Machado
    • 1
  • Fernando Henrique Borges
    • 3
  • Flávia Alessandra Guarnier
    • 2
  • Sara Santos Bernardes
    • 1
  • Júlio Cesar Madureira de-Freitas-Junior
    • 4
  • José Andrés Morgado-Díaz
    • 4
  • Rodrigo Cabral Luiz
    • 1
  • Rubens Cecchini
    • 3
  • Alessandra Lourenço Cecchini
    • 1
  1. 1.Laboratory of Molecular PathologyState University of LondrinaLondrinaBrazil
  2. 2.Laboratory of Pathophysiology and Muscle AdaptationState University of LondrinaLondrinaBrazil
  3. 3.Laboratory of Pathophysiology and Free RadicalsState University of LondrinaLondrinaBrazil
  4. 4.Brazilian National Cancer InstituteINCARio de JaneiroBrazil

Personalised recommendations