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

https://doi.org/10.1016/j.taap.2013.05.010Get rights and content

Highlights

  • Metformin inhibits HO-1 expression in cancer cells.

  • Metformin attenuates Raf-ERK-Nrf2 signaling.

  • Suppression of HO-1 by metformin is independent of AMPK.

  • HO-1 inhibition contributes to anti-proliferative effects of metformin.

Abstract

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.

Introduction

Heme oxygenase-1 (HO-1), as a member of the heat shock protein family, plays a key role as a sensor and regulator of oxidative stress by catalyzing the degradation of heme to form biliverdin, carbon monoxide (CO), and free iron. It plays an important protective role in tissues by reducing oxidative injury, attenuating the inflammatory response, inhibiting apoptosis, and regulating angiogenesis and cell proliferation (Akagi et al., 2005, Wagener et al., 2001). Although it is a cytoprotective enzyme, a growing body of evidence clearly suggests that HO-1 may also play a significant role in the induction of tumorigenic pathways (Jozkowicz et al., 2007, Miyake et al., 2011, Sass et al., 2008). HO-1 is often highly up-regulated in tumor tissues, and its expression is further increased in response to therapy. HO-1 overexpression can inhibit tumor cell apoptosis (Liu et al., 2004) and promote tumor angiogenesis, growth and metastasis (Jozkowicz et al., 2007, Sunamura et al., 2003, Was et al., 2010). Inhibition of HO-1 expression has been suggested as a potential therapeutic approach to sensitization of tumors to chemotherapy and radiotherapy (Alaoui-Jamali et al., 2009, Berberat et al., 2005, Fang et al., 2004).

There is accumulating evidence demonstrating that nuclear erythroid factor 2 (NE-F2)-related factor 2 (Nrf2) is a key transcriptional activator of the antioxidant response element (ARE) that regulates the expression of antioxidant phase II detoxifying enzymes. Interestingly, the promoter region of the HO-1 gene contains an ARE sequence (Kobayashi and Yamamoto, 2005, Kobayashi et al., 2006, Lee and Surh, 2005, Martin et al., 2004). The mechanisms underlying Nrf2 activation are complex, but the available evidence points to two key pathways. The first is a sulfhydryl modification of its cytosolic sequestering protein Keap1 by chemical inducers, which leads to Nrf2 dissociation from Keap1 and subsequent translocation into the nucleus, thereby activating ARE sequences (Kobayashi et al., 2006). In the second pathway, several upstream signaling kinases, including mitogen-activated protein kinases (MAPKs; ERK, p38, and JNK), protein kinase C (PKC), and phosphoinositol 3-kinase (PI3K) regulate Nrf2/ARE activity (Kobayashi and Yamamoto, 2005, Lee and Surh, 2005, Martin et al., 2004). A recent study showed that activated H-Ras promotes transcriptional activation of HO-1 in human renal cancer cells; and H-Ras-induced HO-1 overexpression is mediated primarily through the Raf-ERK activation of Nrf2, which leads to the survival of renal cancer cells (Banerjee et al., 2011).

Metformin (1,1-dimethylbiguanide hydrochloride) is an oral hypoglycemic agent commonly used for the treatment of type 2 diabetes mellitus and nonalcoholic fatty liver disease. Metformin has an excellent therapeutic index with few side effects associated with long-term treatment. Metformin treatment has also been associated with reduced cancer risk. In a study of more than 10,000 diabetic patients being treated with metformin or other sulfonylureas, those that were treated with sulfonylureas had an increased risk of cancer-related mortality when compared to those patients on metformin (Bowker et al., 2006). In a second study using a smaller cohort, patients treated with metformin had a lower incidence of cancer compared to those on other treatments (Evans et al., 2005). Interestingly, this effect appeared to improve with higher doses of metformin. Recent studies show that treatment of diabetics with metformin is associated with a reduced risk of hepatocellular carcinoma and better survival of diabetic patients with pancreatic cancer (Donadon et al., 2009, Donadon et al., 2010, Sadeghi et al., 2012).

Accumulating evidence suggests that metformin has anti-tumor activity. 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. We found that metformin inhibited cancer cell growth by suppressing HO-1 expression through inhibition of a Raf-ERK-Nrf2 signaling and AMPK-independent pathways. This study provides evidence that metformin may be involved in cancer prevention and identifies the mechanisms underlying the reduced cancer risk in diabetic patients treated with this drug.

Section snippets

Reagents and antibodies

Metformin, paclitaxel, zinc protoporphyrin IX (ZnPPIX), and tert-butylhydroquinone (tBHQ) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Compound C and PD98059 were purchased from Calbiochem (La Jolla, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from USB Corp. (Cleveland, OH, USA). The plasmid pCMV-β-gal was purchased from Clontech (Palo Alto, CA, USA). Lipofectamine™ 2000 and SYBR® Safe DNA Gel Stain kit were obtained from Invitrogen

Metformin inhibits HO-1 expression in cancer cells

We first hypothesized that the anti-tumor effects of metformin in cancer cells were due to a decrease in HO-1 expression. Human hepatocellular carcinoma HepG2, lung adenocarcinoma A549 and cervical carcinoma HeLa cell lines were treated with metformin (1–5 mM) at 37 °C for 24 h, and HO-1 protein levels were analyzed by Western blotting. As shown in Fig. 1A, metformin strongly inhibited HO-1 protein expression in a dose-dependent manner, even with constitutive overexpression of HO-1 in A549 cells.

Discussion

Inhibition of HO-1 reduces cancer cell proliferation, tumor growth and increases susceptibility to chemotherapy and radiotherapy in vitro and in vivo (Alaoui-Jamali et al., 2009, Berberat et al., 2005, Fang et al., 2004, Hirai et al., 2007, Nuhn et al., 2009). In the present study, we found that metformin treatment for 24 h strongly suppressed HO-1 expression in hepatocellular carcinoma HepG2 and cervical carcinoma HeLa cell lines (Fig. 1). Interestingly, mutation of Keap1 in non-small cell lung

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgments

This study was supported by a grant (A111580) of the Korean Health Technology R&D Project, Ministry of Health & Welfare, and by a grant (2010-0026220) from National Research Foundation of Korea.

References (47)

  • R. Akagi et al.

    Cytoprotective effects of heme oxygenase in acute renal failure

    Contrib. Nephrol.

    (2005)
  • M.A. Alaoui-Jamali et al.

    A novel experimental heme oxygenase-1-targeted therapy for hormone-refractory prostate cancer

    Cancer Res.

    (2009)
  • I. Ben Sahra et al.

    Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1

    Cancer Res.

    (2011)
  • P.O. Berberat et al.

    Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment

    Clin. Cancer Res.

    (2005)
  • K. Bhalla et al.

    Metformin prevents liver tumorigenesis by inhibiting pathways driving hepatic lipogenesis

    Cancer Prev. Res. (Phila.)

    (2012)
  • S.L. Bowker et al.

    Increased cancer-related mortality for patients with type 2 diabetes who use sulfonylureas or insulin

    Diabetes Care

    (2006)
  • G.M. DeNicola et al.

    Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis

    Nature

    (2011)
  • V. Donadon et al.

    Antidiabetic therapy and increased risk of hepatocellular carcinoma in chronic liver disease

    World J. Gastroenterol.

    (2009)
  • V. Donadon et al.

    Metformin and reduced risk of hepatocellular carcinoma in diabetic patients with chronic liver disease

    Liver Int.

    (2010)
  • J.M. Evans et al.

    Metformin and reduced risk of cancer in diabetic patients

    BMJ

    (2005)
  • J. Fang et al.

    Antiapoptotic role of heme oxygenase (HO) and the potential of HO as a target in anticancer treatment

    Apoptosis

    (2004)
  • K. Hirai et al.

    Inhibition of heme oxygenase-1 by zinc protoporphyrin IX reduces tumor growth of LL/2 lung cancer in C57BL mice

    Int. J. Cancer

    (2007)
  • Y.P. Hwang et al.

    Metformin blocks migration and invasion of tumour cells by inhibition of matrix metalloproteinase-9 activation through a calcium and protein kinase Calpha-dependent pathway: phorbol-12-myristate-13-acetate-induced/extracellular signal-regulated kinase/activator protein-1

    Br. J. Pharmacol.

    (2010)
  • Cited by (103)

    • Oriented nanofibrous P(MMD-co-LA)/Deferoxamine nerve scaffold facilitates peripheral nerve regeneration by regulating macrophage phenotype and revascularization

      2022, Biomaterials
      Citation Excerpt :

      In addition, oxidative stress is another important factor affecting the microenvironment of injured peripheral nerve regeneration [62]. When the NGC is implanted in the damaged nerve site, ischemia, inflammation, and foreign matter reaction will cause a large accumulation of reactive oxygen species (ROS), and the endogenous antioxidant level was not enough to resist the ROS generated at the trauma site, leading to oxidative neural damage [63–65]. Therefore, the restoration of neural activity needs to regulate oxidative attack and inflammatory processes.

    View all citing articles on Scopus
    View full text