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Molecular Diagnosis & Therapy

, Volume 22, Issue 1, pp 115–127 | Cite as

Targeting AMPK, mTOR and β-Catenin by Combined Metformin and Aspirin Therapy in HCC: An Appraisal in Egyptian HCC Patients

  • Doaa Ali Abdelmonsif
  • Ahmed S. Sultan
  • Wessam F. El-Hadidy
  • Dina Mohamed Abdallah
Original Research Article

Abstract

Background

Hepatocellular carcinoma (HCC) is an expanding health problem with a great impact on morbidity and mortality, both in Egypt and worldwide. Recently, metformin and aspirin showed a potential anticancer effect on HCC, although the mechanism of this effect is not fully elucidated.

Objective

The current work aimed to investigate the possibility of targeting AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), and β-catenin proteins through combined metformin/aspirin treatment in the HepG2 cell line, and to explore such molecular targets in Egyptian HCC patients.

Materials and Methods

HepG2 cells were exposed to increasing concentrations of metformin, aspirin and combined treatment, and an MTT assay was performed to determine half maximal inhibitory concentration (IC50). Caspase-3 activity, cell cycle analysis, and protein expression of AMPK, phosphorylated AMPK (pAMPK) and mTOR proteins were assessed. Furthermore, the expression and localization of β-catenin protein was assessed by immunocytochemistry, and protein expression of pAMPK, mTOR and β-catenin was assessed in Egyptian HCC and cirrhotic tissue specimens.

Results

Metformin/aspirin combined treatment had a synergistic effect on cell cycle arrest at the G2/M phase and apoptosis induction in a caspase-dependent manner via downregulation of pAMPK and mTOR protein expression. Additionally, metformin/aspirin combined treatment enhanced cell–cell membrane localization of β-catenin expression in HepG2 cells, which might inhibit the metastatic potential of HepG2 cells. In Egyptian HCC specimens, pAMPK, mTOR and β-catenin proteins showed a significant increased expression compared with cirrhotic controls.

Conclusions

Targeting AMPK, mTOR and β-catenin by combined metformin/aspirin treatment could be a promising therapeutic strategy for Egyptian HCC patients, and possibly other HCC patients.

Notes

Compliance with Ethical Standards

Conflict of interest

Doaa Ali Abdelmonsif, Ahmed S. Sultan, Wessam F. EL-Hadidy and Dina Mohamed Abdallah have read the journal’s policy on conflicts of interest and disclose no conflicts.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Alexandria Faculty of Medicine Research Committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Since the current study is retrospective, formal consent was not required.

References

  1. 1.
    Ge S, Huang D. Systemic therapies for hepatocellular carcinoma. Drug Discov Ther. 2015;9(5):352–62.CrossRefPubMedGoogle Scholar
  2. 2.
    Bellissimo F, Pinzone MR, Cacopardo B, Nunnari G. Diagnostic and therapeutic management of hepatocellularcarcinoma. World J Gastroenterol. 2015;21(42):12003–21.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Sabry Ael H, El-Aal AA, Mahmoud NS, Nabil Y, Aziz IZ. An initial indication of predisposing risk of schistosoma mansoni infection for hepatocellular carcinoma. J Egypt Soc Parasitol. 2015;45(2):233–40.CrossRefPubMedGoogle Scholar
  4. 4.
    European Association for the Study of the Liver, European Organisation for Research and Treatment of Cancer. EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol. 2012;56(4):908–43.Google Scholar
  5. 5.
    Facciorusso A, Licinio R, Carr BI, Di Leo A, Barone M. MEK 1/2 inhibitors in the treatment of hepatocellular carcinoma. Expert Rev Gastroenterol Hepatol. 2015;9(7):993–1003.CrossRefPubMedGoogle Scholar
  6. 6.
    Li L, Wang H. Heterogeneity of liver cancer and personalized therapy. Cancer Lett. 2016;379(2):191–7.CrossRefPubMedGoogle Scholar
  7. 7.
    Carling D, Sanders MJ, Woods A. The regulation of AMP-activated protein kinase by upstream kinases. Int J Obes (Lond). 2008;32(Suppl 4):S55–9.CrossRefGoogle Scholar
  8. 8.
    Viollet B, Guigas B, Leclerc J, Hebrard S, Lantier L, Mounier R, et al. AMP-activated protein kinase in the regulation of hepatic energy metabolism: from physiology to therapeutic perspectives. Acta Physiol (Oxford). 2009;196(1):81–98.CrossRefGoogle Scholar
  9. 9.
    Ishijima N, Kanki K, Shimizu H, Shiota G. Activation of AMP-activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib. Cancer Sci. 2015;106(5):567–75.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zadra G, Batista JL, Loda M. Dissecting the dual role of AMPK in cancer: from experimental to human studies. Mol Cancer Res. 2015;13(7):1059.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Laplante M, Sabatini DM. mTOR signaling ingrowth control and disease. Cell. 2012;149(2):274–93.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Liko D, Hall MN. mTOR in health and in sickness. J Mol Med (Berlin). 2015;93(10):1061–73.CrossRefGoogle Scholar
  13. 13.
    Matter MS, Decaens T, Andersen JB, Thorgeirsson SS. Targeting the mTOR pathway in hepatocellular carcinoma: current state and future trends. J Hepatol. 2014;60(4):855–65.CrossRefPubMedGoogle Scholar
  14. 14.
    Cholongitas E, Mamou C, Rodriguez-Castro KI, Burra P. Mammalian target of rapamycin inhibitors are associated with lower rates of hepatocellular carcinoma recurrence after liver transplantation: a systematic review. Transpl Int. 2014;27(10):1039–49.CrossRefPubMedGoogle Scholar
  15. 15.
    Ferrín G, Aguilar-Melero P, Rodríguez-Perálvarez M, Montero-Álvarez JL, de la Mata M. Biomarkers for hepatocellular carcinoma: diagnostic and therapeutic utility. Hepatic Med. 2015;7:1–10.Google Scholar
  16. 16.
    Anastas JN, Moon RT. WNT signalling pathways as therapeutic targets in cancer. Nat Rev Cancer. 2013;13(1):11–26.CrossRefPubMedGoogle Scholar
  17. 17.
    White BD, Chien AJ, Dawson DW. Dysregulation of Wnt/beta-catenin signaling in gastrointestinal cancers. Gastroenterology. 2012;142(2):219–32.CrossRefPubMedGoogle Scholar
  18. 18.
    Lai TY, Su CC, Kuo WW, Yeh YL, Kuo WH, Tsai FJ, et al. β-Catenin plays a key role in metastasis of human hepatocellular carcinoma. Oncol Rep. 2011;26(2):415–22.PubMedGoogle Scholar
  19. 19.
    Vilchez V, Turcios L, Marti F, Gedaly R. Targeting Wnt/β-catenin pathway in hepatocellular carcinoma treatment. World J Gastroenterol. 2016;22(2):823–32.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wang J, Gao Q, Wang D, Wang Z, Hu C. Metformin inhibits growth of lung adenocarcinoma cells by inducing apoptosis via the mitochondria-mediated pathway. Oncol Lett. 2015;10(3):1343–9.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Zhou X, Chen J, Yi G, DengM Liu H, Liang M, et al. Metformin suppresses hypoxia-induced stabilization of HIF-1alpha through reprogramming of oxygen metabolism in hepatocellular carcinoma. Oncotarget. 2016;7(1):873–84.PubMedGoogle Scholar
  22. 22.
    Alfonso L, Ai G, Spitale RC, Bhat GJ. Molecular targets of aspirin and cancer prevention. Br J Cancer. 2014;111(1):61–7.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Sahasrabuddhe VV, Gunja MZ, Graubard BI, Trabert B, Schwartz LM, Park Y, et al. Nonsteroidal anti-inflammatory drug use, chronic liver disease, and hepatocellular carcinoma. J Natl Cancer Inst. 2012;104(23):1808–14.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Veitonmaki T, Tammela TL, Auvinen A, Murtola TJ. Use of aspirin, but not other non-steroidal anti-inflammatory drugs is associated with decreased prostate cancer risk at the population level. Eur J Cancer. 2013;49(4):938–45.CrossRefPubMedGoogle Scholar
  25. 25.
    Din FV, Valanciute A, Houde VP, Zibrova D, Green KA, Sakamoto K, et al. Aspirin inhibits mTOR signaling, activates AMP-activated protein kinase, and induces autophagy in colorectal cancer cells. Gastroenterology. 2012;142(7):1504–15.e3.Google Scholar
  26. 26.
    Edmondson HA, Steiner PE. Primary carcinomaof the liver: a study of 100 cases among 48,900 necropsies. Cancer. 1954;7(3):462–503.CrossRefPubMedGoogle Scholar
  27. 27.
    Leal P, Garcia P, Sandoval A, Letelier P, Brebi P, Ili C, et al. Immunohistochemical expression of phospho-mTOR is associated with poor prognosis inpatients with gallbladder adenocarcinoma. Arch Pathol Lab Med. 2013;137(4):552–7.CrossRefPubMedGoogle Scholar
  28. 28.
    Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E, et al. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol. 1999;52(6):455–60.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Bonifazi M, Gallus S, Bosetti C, Polesel J, Serraino D, Talamini R, et al. Aspirin use and pancreatic cancer risk. Eur J Cancer Prev. 2010;19(5):352–4.CrossRefPubMedGoogle Scholar
  30. 30.
    Sahin IH, Hassan MM, Garrett CR. Impact of non-steroidal anti-inflammatory drugs on gastrointestinal cancers: current state-of-the science. Cancer Lett. 2014;345(2):249–57.CrossRefPubMedGoogle Scholar
  31. 31.
    Dachineni R, Ai G, Kumar DR, Sadhu SS, Tummala H, Bhat GJ. Cyclin A2 and CDK2 as novel targets of aspirin and salicylic acid: a potential role in cancer prevention. Mol Cancer Res. 2016;14(3):241–52.CrossRefPubMedGoogle Scholar
  32. 32.
    Ai G, Dachineni R, Muley P, Tummala H, Bhat GJ. Aspirin and salicylic acid decrease c-Myc expression in cancer cells: a potential role in chemoprevention. Tumour Biol. 2016;37(2):1727–38.CrossRefPubMedGoogle Scholar
  33. 33.
    Wen L, Liang C, Chen E, Chen W, Liang F, Zhi X, et al. Regulation of multi-drug resistance in hepatocellular carcinoma cells is TRPC6/calcium dependent. Sci Rep. 2016;6:23269.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Din FVN, Dunlop MG, Stark LA. Evidence for colorectal cancer cell specificity of aspirin effects on NFκB signalling and apoptosis. Br J Cancer. 2004;91(2):381–8.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Yue W, Zheng X, Lin Y, Yang CS, Xu Q, Carpizo D, et al. Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2. Oncotarget. 2015;6(25):21208–24.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Park HU, Suy S, Danner M, Dailey V, Zhang Y, Li H, et al. AMP-activated protein kinase promotes human prostate cancer cell growth and survival. Mol Cancer Ther. 2009;8(4):733–41.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Zheng L, Yang W, Wu F, Wang C, Yu L, Tang L, et al. Prognostic significance of AMPK activation and therapeutic effects of metformin in hepatocellular carcinoma. Clin Cancer Res. 2013;19(19):5372–80.CrossRefPubMedGoogle Scholar
  38. 38.
    Hardie DG, Alessi DR. LKB1 and AMPK and the cancer-metabolism link: 10 years after. BMC Biol. 2013;11(1):36.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Faubert B, Vincent EE, Poffenberger MC, Jones RG. The AMP-activated protein kinase (AMPK) and cancer: many faces of a metabolic regulator. Cancer Lett. 2015;356(2 Pt A):165–70.CrossRefPubMedGoogle Scholar
  40. 40.
    Laderoute K, Calaoagan JM, Madrid PB, Klon AE, Ehrlich PJ. SU11248 (sunitinib) directly inhibits the activity of mammalian 5′-AMP-activated protein kinase (AMPK). Cancer Biol Ther. 2010;10(1):68–76.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Motoshima H, Goldstein BJ, Igata M, Araki E. AMPK and cell proliferation—AMPK as a therapeutic target for atherosclerosis and cancer. J Physiol. 2006;574(Pt 1):63–71.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Luo Z, Zang M, Guo W. AMPK as a metabolic tumor suppressor: control of metabolism and cell growth. Future Oncol. 2010;6(3):457–70.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Sun Y, Tao C, Huang X, He H, Shi H, Zhang Q, et al. Metformin induces apoptosis of human hepatocellular carcinoma HepG2 cells by activating an AMPK/p53/miR-23a/FOXA1 pathway. OncoTargets and Ther. 2016;9:2845–53.Google Scholar
  44. 44.
    Jo W, Yu ES, Chang M, Park HK, Choi HJ, Ryu JE, et al. Metformin inhibits early stage diethylnitrosamine induced hepatocarcinogenesis in rats. Mol Med Rep. 2016;13(1):146–52.CrossRefPubMedGoogle Scholar
  45. 45.
    Faubert B, Vincent EE, Poffenberger MC, Jones RG. The AMP-activated protein kinase (AMPK) and cancer: many faces of a metabolic regulator. Cancer Lett. 2015;356(2):165–70.CrossRefPubMedGoogle Scholar
  46. 46.
    Sieghart W, Fuereder T, Schmid K, Cejka D, Werzowa J, Wrba F, et al. Mammalian target of rapamycin pathway activity in hepatocellular carcinomas of patients undergoing liver transplantation. Transplantation. 2007;83(4):425–32.CrossRefPubMedGoogle Scholar
  47. 47.
    Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C, et al. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology. 2008;135(6):1972–83 (83.e1–11).CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Tam KH, Yang ZF, Lau CK, Lam CT, Pang RW, Poon RT. Inhibition of mTOR enhances chemosensitivity in hepatocellular carcinoma. Cancer Lett. 2009;273(2):201–9.CrossRefPubMedGoogle Scholar
  49. 49.
    Ben Sahra I, Regazzetti C, Robert G, Laurent K, Le Marchand-Brustel Y, Auberger P, et al. Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1. Cancer Res. 2011;71(13):4366–72.CrossRefPubMedGoogle Scholar
  50. 50.
    Kalender A, Selvaraj A, Kim SY, Gulati P, Brule S, Viollet B, et al. Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner. Cell Metab. 2010;11(5):390–401.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    O’Brien AJ, Villani LA, Broadfield LA, Houde VP, Galic S, Blandino G, et al. Salicylate activates AMPK and synergizes with metformin to reduce the survival of prostate and lung cancer cells ex vivo through inhibition of de novo lipogenesis. Biochem J. 2015;469(2):177–87.CrossRefPubMedGoogle Scholar
  52. 52.
    Marimuthu S, Chivukula RS, Alfonso LF, Moridani M, Hagen FK, Bhat GJ. Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: identification of novel targets. Int J Oncol. 2011;39(5):1273–83.PubMedGoogle Scholar
  53. 53.
    Ai G, Dachineni R, Kumar DR, Alfonso LF, Marimuthu S, Bhat GJ. Aspirin inhibits glucose6phosphate dehydrogenase activity in HCT 116 cells through acetylation: identification of aspirin-acetylated sites. Mol Med Rep. 2016;14(2):1726–32.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Memmott RM, Mercado JR, Maier CR, Kawabata S, Fox SD, Dennis PA. Metformin prevents tobacco carcinogen-induced lung tumorigenesis. Cancer Prev Res (Philadelphia, PA). 2010;3(9):1066–76.CrossRefGoogle Scholar
  55. 55.
    Kim EH, Kim M-S, Cho C-K, Jung W-G, Jeong YK, Jeong J-H. Low and high linear energy transfer radiation sensitization of HCC cells by metformin. J Radiat Res. 2014;55(3):432–42.CrossRefPubMedGoogle Scholar
  56. 56.
    Cheng J, Huang T, Li Y, Guo Y, Zhu Y, Wang Q, et al. AMP-activated protein kinase suppresses the in vitro and in vivo proliferation of hepatocellular carcinoma. PLoS One. 2014;9(4):e93256.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Cai X, Hu X, Cai B, Wang Q, Li Y, Tan X, et al. Metformin suppresses hepatocellular carcinoma cell growth through induction of cell cycle G1/G0 phase arrest and p21CIP and p27KIP expression and downregulation of cyclin D1 in vitro and in vivo. Oncol Rep. 2013;30(5):2449–57.CrossRefPubMedGoogle Scholar
  58. 58.
    Gao L, Williams JL. Nitric oxide-donating aspirin inducesG2/M phase cell cycle arrest in human cancer cells by regulating phase transition proteins. Int J Oncol. 2012;41(1):325–30.PubMedPubMedCentralGoogle Scholar
  59. 59.
    Tewari D, Majumdar D, Vallabhaneni S, Bera AK. Aspirin induces cell death by directly modulating mitochondrial voltage-dependent anion channel (VDAC). Sci Rep. 2017;7:45184.CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Bos CL, Kodach LL, van den Brink GR, Diks SH, van Santen MM, Richel DJ, et al. Effect of aspirin on the Wnt//[beta]-catenin pathway is mediated via protein phosphatase 2A. Oncogene. 2006;25(49):6447–56.CrossRefPubMedGoogle Scholar
  61. 61.
    Subramaniam N, Sherman MH, Rao R, Wilson C, Coulter S, Atkins AR, et al. Metformin-mediated bambi expression in hepatic stellate cells induces prosurvival Wnt/β-catenin signaling. Cancer Prev Res. 2012;5(4):553–61.CrossRefGoogle Scholar
  62. 62.
    Zekri AR, Youssef AS, Bakr YM, Gabr RM, El-Rouby MN, Hammad I, et al. Serum biomarkers for early detection of hepatocellular carcinoma associated with HCV infection in egyptian patients. Asian Pac J Cancer Prev. 2015;16(3):1281–7.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.Department of Medical Biochemistry, Faculty of MedicineAlexandria UniversityAlexandriaEgypt
  2. 2.Molecular Biology and Nanomedicine Laboratories, Centre of Excellence for Regenerative Medicine Research and Applications, Faculty of MedicineAlexandria UniversityAlexandriaEgypt
  3. 3.Department of Biochemistry, Faculty of ScienceAlexandria UniversityAlexandriaEgypt
  4. 4.Department of Pharmacology and Experimental Therapeutics, Medical Research InstituteAlexandria UniversityAlexandriaEgypt
  5. 5.Department of Pathology, Faculty of MedicineAlexandria UniversityAlexandriaEgypt

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