Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf-ERK-Nrf2 signaling and AMPK-independent pathways

Toxicol Appl Pharmacol. 2013 Sep 1;271(2):229-38. doi: 10.1016/j.taap.2013.05.010. Epub 2013 May 21.


Resistance to therapy is the major obstacle to more effective cancer treatment. Heme oxygenase-1 (HO-1) is often highly up-regulated in tumor tissues, and its expression is further increased in response to therapies. It has been suggested that inhibition of HO-1 expression is a potential therapeutic approach to sensitize tumors to chemotherapy and radiotherapy. In this study, we tested the hypothesis that the anti-tumor effects of metformin are mediated by suppression of HO-1 expression in cancer cells. Our results indicate that metformin strongly suppresses HO-1 mRNA and protein expression in human hepatic carcinoma HepG2, cervical cancer HeLa, and non-small-cell lung cancer A549 cells. Metformin also markedly reduced Nrf2 mRNA and protein levels in whole cell lysates and suppressed tert-butylhydroquinone (tBHQ)-induced Nrf2 protein stability and antioxidant response element (ARE)-luciferase activity in HepG2 cells. We also found that metformin regulation of Nrf2 expression is mediated by a Keap1-independent mechanism and that metformin significantly attenuated Raf-ERK signaling to suppress Nrf2 expression in cancer cells. Inhibition of Raf-ERK signaling by PD98059 decreased Nrf2 mRNA expression in HepG2 cells, confirming that the inhibition of Nrf2 expression is mediated by an attenuation of Raf-ERK signaling in cancer cells. The inactivation of AMPK by siRNA, DN-AMPK or the pharmacological AMPK inhibitor compound C, revealed that metformin reduced HO-1 expression in an AMPK-independent manner. These results highlight the Raf-ERK-Nrf2 axis as a new molecular target in anticancer therapy in response to metformin treatment.

Keywords: AMP-activated protein kinase; AMPK; ARE; ERK; HO-1; Keap1; Kelch-like ECH-associated protein 1; Metformin; Nrf2; antioxidant response element; extracellular signal-regulated kinase; heme oxygenase-1; nuclear erythroid factor 2 (NE-F2)-related factor 2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cell Line, Tumor
  • Cell Nucleus / chemistry
  • Cell Survival / drug effects
  • Cytosol / chemistry
  • Galactosidases / metabolism
  • Heme Oxygenase-1 / antagonists & inhibitors*
  • Humans
  • Hypoglycemic Agents / pharmacology*
  • Indicators and Reagents
  • Luciferases / metabolism
  • MAP Kinase Signaling System / drug effects*
  • Metformin / pharmacology*
  • Mitogen-Activated Protein Kinases / drug effects*
  • NF-E2-Related Factor 2 / physiology*
  • Neoplasms / enzymology*
  • Plasmids / genetics
  • RNA / biosynthesis
  • RNA / genetics
  • RNA, Small Interfering
  • Real-Time Polymerase Chain Reaction
  • Signal Transduction / drug effects
  • Transfection
  • raf Kinases / drug effects*


  • Hypoglycemic Agents
  • Indicators and Reagents
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • RNA, Small Interfering
  • RNA
  • Metformin
  • Luciferases
  • Heme Oxygenase-1
  • raf Kinases
  • Mitogen-Activated Protein Kinases
  • Galactosidases