Tumor Biology

, Volume 37, Issue 8, pp 11199–11208 | Cite as

Metformin and AICAR regulate NANOG expression via the JNK pathway in HepG2 cells independently of AMPK

  • Chen Shen
  • Sun-O Ka
  • Su Jin Kim
  • Ji Hye Kim
  • Byung-Hyun Park
  • Ji Hyun Park
Original Article


NANOG, a marker of stemness, impacts tumor progression and therapeutic resistance in cancer cells. In human hepatocellular carcinoma (HCC), upregulation of NANOG is associated with metastasis and a low survival rate, while its downregulation results in a lower colony formation rate and enhanced chemosensitivity. Metformin, an agent widely used for diabetes treatment, and AICAR, another AMP-activated protein kinase (AMPK) activator, have been reported to inhibit the growth of several types of cancer. Although inhibitory effects of metformin on NANOG in pancreatic cancer cells and of AICAR in mouse embryonic stem cells have been described, the underlying molecular mechanisms remain uncertain in HCC. In this study, we used the HepG2 cell line and found that metformin/AICAR downregulated NANOG expression with decreased cell viability and enhanced chemosensitivity to 5-fluorouracil (5-FU). Moreover, metformin/AICAR inhibited c-Jun N-terminal kinase (JNK) activity, and blockade of either the JNK MAPK pathway or knockdown of JNK1 gene expression reduced NANOG levels. The upregulation of NANOG and phospho-JNK by basic fibroblast growth factor (bFGF) was abrogated by metformin/AICAR. Additionally, although transient upregulation of NANOG within 2 h of treatment with metformin/AICAR was concordant with both JNK and AMPK activation, increased NANOG expression with activation of JNK was also observed following AMPK inhibition with compound C. Taken together, our data suggest that metformin/AICAR regulate NANOG expression via the JNK MAPK pathway in HepG2 cells independently of AMPK, and that this JNK/NANOG signaling pathway may offer new therapeutic strategies for the treatment of HCC.





This work was supported by research funds from the Biomedical Research Institute of Chonbuk National University Hospital.

Compliance with ethical standards

Conflicts of interest



  1. 1.
    Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108.CrossRefPubMedGoogle Scholar
  2. 2.
    Hsieh SC, Tsai JP, Yang SF, Tang MJ, Hsieh YH. Metformin inhibits the invasion of human hepatocellular carcinoma cells and enhances the chemosensitivity to sorafenib through a downregulation of the erk/jnk-mediated nf-kappab-dependent pathway that reduces upa and mmp-9 expression. Amino Acids. 2014;46:2809–22.CrossRefPubMedGoogle Scholar
  3. 3.
    Miyoshi H, Kato K, Iwama H, Maeda E, Sakamoto T, Fujita K, et al. Effect of the anti-diabetic drug metformin in hepatocellular carcinoma in vitro and in vivo. Int J Oncol. 2014;45:322–32.PubMedGoogle Scholar
  4. 4.
    Sauer H, Engel S, Milosevic N, Sharifpanah F, Wartenberg M. Activation of AMP-kinase by AICAR induces apoptosis of DU-145 prostate cancer cells through generation of reactive oxygen species and activation of c-jun N-terminal kinase. Int J Oncol. 2012;40:501–8.PubMedGoogle Scholar
  5. 5.
    Jose C, Hebert-Chatelain E, Bellance N, Larendra A, Su M, Nouette-Gaulain K, et al. AICAR inhibits cancer cell growth and triggers cell-type distinct effects on OXPHOS biogenesis, oxidative stress and Akt activation. Biochim Biophys Acta. 1807;2011:707–18.Google Scholar
  6. 6.
    Sui X, Xu Y, Yang J, Fang Y, Lou H, Han W, et al. Use of metformin alone is not associated with survival outcomes of colorectal cancer cell but AMPK activator AICAR sensitizes anticancer effect of 5-fluorouracil through AMPK activation. PLoS ONE. 2014;9:e97781.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wu Y, Qi Y, Liu H, Wang X, Zhu H, Wang Z. AMPK activator AICAR promotes 5-FU-induced apoptosis in gastric cancer cells. Mol Cell Biochem. 2016;411:299–305.CrossRefPubMedGoogle Scholar
  8. 8.
    Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318:1917–20.CrossRefPubMedGoogle Scholar
  9. 9.
    Gong S, Li Q, Jeter CR, Fan Q, Tang DG, Liu B. Regulation of NANOG in cancer cells. Mol Carcinog. 2015;54:679–87.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Sun C, Sun L, Jiang K, Gao DM, Kang XN, Wang C, et al. NANOG promotes liver cancer cell invasion by inducing epithelial-mesenchymal transition through NODAL/SMAD3 signaling pathway. Int J Biochem Cell Biol. 2013;45:1099–108.CrossRefPubMedGoogle Scholar
  11. 11.
    Zhou JJ, Deng XG, He XY, Zhou Y, Yu M, Gao WC, et al. Knockdown of NANOG enhances chemosensitivity of liver cancer cells to doxorubicin by reducing MDR1 expression. Int J Oncol. 2014;44:2034–40.PubMedGoogle Scholar
  12. 12.
    Bao B, Wang Z, Ali S, Ahmad A, Azmi AS, Sarkar SH, et al. Metformin inhibits cell proliferation, migration and invasion by attenuating CSC function mediated by deregulating miRNAs in pancreatic cancer cells. Cancer Prev Res. 2012;5:355–64.CrossRefGoogle Scholar
  13. 13.
    Chae HD, Lee MR, Broxmeyer HE. 5-Aminoimidazole-4-carboxyamide ribonucleoside induces g(1)/s arrest and NANOG downregulation via p53 and enhances erythroid differentiation. Stem Cells. 2012;30:140–9.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Wang YP, Yu GR, Lee MJ, Lee SY, Chu IS, Leem SH, et al. Lipocalin-2 negatively modulates the epithelial-to-mesenchymal transition in hepatocellular carcinoma through the epidermal growth factor (TGF-beta1)/lcn2/twist1 pathway. Hepatology. 2013;58:1349–61.CrossRefPubMedGoogle Scholar
  15. 15.
    Risal P, Hwang PH, Yun BS, Yi HK, Cho BH, Jang KY, et al. Hispidin analogue davallialactone attenuates carbon tetrachloride-induced hepatotoxicity in mice. J Nat Prod. 2012;75:1683–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Cadena-Herrera D, Esparza-De Lara JE, Ramírez-Ibañez ND, López-Morales CA, Pérez NO, Flores-Ortiz LF, et al. Validation of three viable-cell counting methods: manual, semi-automated, and automated. Biotechnol Reprod. 2015;7:9–16.CrossRefGoogle Scholar
  17. 17.
    Rattan R, Giri S, Singh AK, Singh I. 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase. J Biol Chem. 2005;280:39582–93.CrossRefPubMedGoogle Scholar
  18. 18.
    Xu G, Ji W, Su Y, Xu Y, Yan Y, Shen S, et al. Sulfatase 1 (hSulf-1) reverses basic fibroblast growth factor-stimulated signaling and inhibits growth of hepatocellular carcinoma in animal model. Oncotarget. 2014;5:5029–39.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Sukarawan W, Nowwarote N, Kerdpon P, Pavasant P, Osathanon T. Effect of basic fibroblast growth factor on pluripotent marker expression and colony forming unit capacity of stem cells isolated from human exfoliated deciduous teeth. Odontology. 2014;102:160–6.CrossRefPubMedGoogle Scholar
  20. 20.
    Meisse D, Van de Casteele M, Beauloye C, Hainault I, Kefas BA, Rider MH, et al. Sustained activation of AMP-activated protein kinase induces c-jun N-terminal kinase activation and apoptosis in liver cells. FEBS Lett. 2002;526:38–42.CrossRefPubMedGoogle Scholar
  21. 21.
    Wu N, Gu C, Gu H, Hu H, Han Y, Li Q. Metformin induces apoptosis of lung cancer cells through activating JNK/p38 MAPK pathway and GADD153. Neoplasma. 2011;58:482–90.CrossRefPubMedGoogle Scholar
  22. 22.
    Marinello PC, da Silva TN, Panis C, Neves AF, Machado KL, Borges FH, Guarnier FA, Bernardes SS, de-Freitas-Junior JC, Morgado-Diaz JA, Luiz RC, Cecchini R, Cecchini AL. 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 beta1 induction. Tumour Biol. 2015.Google Scholar
  23. 23.
    Yang X, Sun D, Tian Y, Ling S, Wang L. Metformin sensitizes hepatocellular carcinoma to arsenic trioxide-induced apoptosis by downregulating bcl2 expression. Tumour Biol. 2015;36:2957–64.CrossRefPubMedGoogle Scholar
  24. 24.
    Lee JH, Kim JH, Kim JS, Chang JW, Kim SB, Park JS, et al. AMP-activated protein kinase inhibits TGF-beta-, angiotensin II-, aldosterone-, high glucose-, and albumin-induced epithelial-mesenchymal transition. Am J Physiol Renal Physiol. 2013;304:F686–97.CrossRefPubMedGoogle Scholar
  25. 25.
    Chen X, Hu C, Zhang W, Shen Y, Wang J, Hu F, et al. Metformin inhibits the proliferation, metastasis, and cancer stem-like sphere formation in osteosarcoma MG63 cells in vitro. Tumour Biol. 2015;36:9873–83.CrossRefPubMedGoogle Scholar
  26. 26.
    Gou S, Cui P, Li X, Shi P, Liu T, Wang C. Low concentrations of metformin selectively inhibit CD133(+) cell proliferation in pancreatic cancer and have anticancer action. PLoS ONE. 2013;8:e63969.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 2003;113:643–55.CrossRefPubMedGoogle Scholar
  28. 28.
    Shan J, Shen J, Liu L, Xia F, Xu C, Duan G, et al. Nanog regulates self-renewal of cancer stem cells through the insulin-like growth factor pathway in human hepatocellular carcinoma. Hepatology. 2012;56:1004–14.CrossRefPubMedGoogle Scholar
  29. 29.
    Yin X, Zhang BH, Zheng SS, Gao DM, Qiu SJ, Wu WZ, et al. Coexpression of gene Oct4 and Nanog initiates stem cell characteristics in hepatocellular carcinoma and promotes epithelial-mesenchymal transition through activation of Stat3/Snail signaling. J Hematol Oncol. 2015;8:23.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Sid B, Glorieux C, Valenzuela M, Rommelaere G, Najimi M, Dejeans N, et al. AICAR induces Nrf2 activation by an AMPK-independent mechanism in hepatocarcinoma cells. Biochem Pharmacol. 2014;91:168–80.CrossRefPubMedGoogle Scholar
  31. 31.
    Bogoyevitch MA, Kobe B. Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases. Microbiol Mol Biol Rev. 2006;70:1061–95.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Guo L, Guo Y, Xiao S, Shi X. Protein kinase p-JNK is correlated with the activation of AP-1 and its associated Jun family proteins in hepatocellular carcinoma. Life Sci. 2005;77:1869–78.CrossRefPubMedGoogle Scholar
  33. 33.
    Tong M, Fung TM, Luk ST, Ng KY, Lee TK, Lin CH, et al. ANXA3/JNK signaling promotes self-renewal and tumor growth, and its blockade provides a therapeutic target for hepatocellular carcinoma. Stem Cell Rep. 2015;5:45–59.CrossRefGoogle Scholar
  34. 34.
    Brill LM, Xiong W, Lee KB, Ficarro SB, Crain A, Xu Y, et al. Phosphoproteomic analysis of human embryonic stem cells. Cell Stem Cell. 2009;5:204–13.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001;108:1167–74.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Dai RY, Zhao XF, Li JJ, Chen R, Luo ZL, Yu LX, et al. Implication of transcriptional repression in compound C-induced apoptosis in cancer cells. Cell Death Dis. 2013;4:e883.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Liu X, Chhipa RR, Nakano I, Dasgupta B. The AMPK inhibitor compound C is a potent AMPK-independent antiglioma agent. Mol Cancer Ther. 2014;13:596–605.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Chen Shen
    • 1
  • Sun-O Ka
    • 2
  • Su Jin Kim
    • 1
  • Ji Hye Kim
    • 3
  • Byung-Hyun Park
    • 2
  • Ji Hyun Park
    • 1
    • 4
  1. 1.Division of Endocrinology and Metabolism, Department of Internal Medicine, Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University HospitalChonbuk National University Medical SchoolJeonjuSouth Korea
  2. 2.Department of BiochemistryChonbuk National University Medical SchoolJeonjuSouth Korea
  3. 3.Division of Endocrinology and Metabolism, Department of Internal MedicinePresbyterian Medical CenterJeonjuSouth Korea
  4. 4.Division of Endocrinology and Metabolism, Department of Internal MedicineChonbuk National University Medical SchoolJeonjuSouth Korea

Personalised recommendations