Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf-ERK-Nrf2 signaling and AMPK-independent pathways
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)
- et al.
The heme oxygenase-1 protein is overexpressed in human renal cancer cells following activation of the Ras-Raf-ERK pathway and mediates anti-apoptotic signal
J. Biol. Chem.
(2011) - et al.
Therapeutic strategies by modulating oxygen stress in cancer and inflammation
Adv. Drug Deliv. Rev.
(2009) - et al.
Constitutive overexpression of Nrf2-dependent heme oxygenase-1 in A549 cells contributes to resistance to apoptosis induced by epigallocatechin 3-gallate
J. Biol. Chem.
(2006) - et al.
Nrf2 as a novel molecular target for chemoprevention
Cancer Lett.
(2005) - et al.
AMP-activated protein kinase activity is critical for hypoxia-inducible factor-1 transcriptional activity and its target gene expression under hypoxic conditions in DU145 cells
J. Biol. Chem.
(2003) - et al.
Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol
J. Biol. Chem.
(2004) - et al.
Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome
J. Biol. Chem.
(2003) - et al.
Luteolin inhibits Nrf2 leading to negative regulation of the Nrf2/ARE pathway and sensitization of human lung carcinoma A549 cells to therapeutic drugs
Free Radic. Biol. Med.
(2011) - et al.
Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase
Blood
(2001) - et al.
Metformin impairs the growth of liver kinase B1-intact cervical cancer cells
Gynecol. Oncol.
(2012)
Cytoprotective effects of heme oxygenase in acute renal failure
Contrib. Nephrol.
A novel experimental heme oxygenase-1-targeted therapy for hormone-refractory prostate cancer
Cancer Res.
Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1
Cancer Res.
Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment
Clin. Cancer Res.
Metformin prevents liver tumorigenesis by inhibiting pathways driving hepatic lipogenesis
Cancer Prev. Res. (Phila.)
Increased cancer-related mortality for patients with type 2 diabetes who use sulfonylureas or insulin
Diabetes Care
Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis
Nature
Antidiabetic therapy and increased risk of hepatocellular carcinoma in chronic liver disease
World J. Gastroenterol.
Metformin and reduced risk of hepatocellular carcinoma in diabetic patients with chronic liver disease
Liver Int.
Metformin and reduced risk of cancer in diabetic patients
BMJ
Antiapoptotic role of heme oxygenase (HO) and the potential of HO as a target in anticancer treatment
Apoptosis
Inhibition of heme oxygenase-1 by zinc protoporphyrin IX reduces tumor growth of LL/2 lung cancer in C57BL mice
Int. J. Cancer
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.
Cited by (103)
Targeting the NRF2/KEAP1 pathway in cervical and endometrial cancers
2023, European Journal of PharmacologyHeme oxygenase-1 & 2 and their potential contribution in heme induced colorectal carcinogenesis
2022, Pathology Research and PracticeOriented nanofibrous P(MMD-co-LA)/Deferoxamine nerve scaffold facilitates peripheral nerve regeneration by regulating macrophage phenotype and revascularization
2022, BiomaterialsCitation 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.
Biguanides drugs: Past success stories and promising future for drug discovery
2021, European Journal of Medicinal Chemistry