Copper Ion from Cu2O Crystal Induces AMPK-Mediated Autophagy via Superoxide in Endothelial Cells

  • Seo, Youngsik (Department of Molecular Biology & Institute of Nanosensor and Biotechnology, Dankook University) ;
  • Cho, Young-Sik (Department of Chemistry, Dankook University) ;
  • Huh, Young-Duk (Department of Chemistry, Dankook University) ;
  • Park, Heonyong (Department of Molecular Biology & Institute of Nanosensor and Biotechnology, Dankook University)
  • Received : 2015.07.14
  • Accepted : 2015.09.19
  • Published : 2016.03.31


Copper is an essential element required for a variety of functions exerted by cuproproteins. An alteration of the copper level is associated with multiple pathological conditions including chronic ischemia, atherosclerosis and cancers. Therefore, copper homeostasis, maintained by a combination of two copper ions ($Cu^+$ and $Cu^{2+}$), is critical for health. However, less is known about which of the two copper ions is more toxic or functional in endothelial cells. Cubic-shaped $Cu_2O$ and CuO crystals were prepared to test the role of the two different ions, $Cu^+$ and $Cu^{2+}$, respectively. The $Cu_2O$ crystal was found to have an effect on cell death in endothelial cells whereas CuO had no effect. The $Cu_2O$ crystals appeared to induce p62 degradation, LC3 processing and an elevation of LC3 puncta, important processes for autophagy, but had no effect on apoptosis and necrosis. $Cu_2O$ crystals promote endothelial cell death via autophagy, elevate the level of reactive oxygen species such as superoxide and nitric oxide, and subsequently activate AMP-activated protein kinase (AMPK) through superoxide rather than nitric oxide. Consistently, the AMPK inhibitor Compound C was found to inhibit $Cu_2O$-induced AMPK activation, p62 degradation, and LC3 processing. This study provides insight on the pathophysiologic function of $Cu^+$ ions in the vascular system, where $Cu^+$ induces autophagy while $Cu^{2+}$ has no detected effect.


Supported by : Dankook University


  1. Abello, P.A., Fidler, S.A., Bulkley, G.B., and Buchman, T.G. (1994). Antioxidants modulate induction of programmed endothelial cell death (apoptosis) by endotoxin. Arch. Surg. 129, 134-140.
  2. Ahn, S., Park, J., An, I., Jung, S.J., and Hwang, J. (2014). Transient receptor potential cation channel V1 (TRPV1) is degraded by starvation- and glucocorticoid-mediated autophagy. Mol. Cells 37, 257-263.
  3. Altekin, E., Coker, C., Sişman, A.R., Onvural, B., Kuralay, F., and Kirimli, O. (2005). The relationship between trace elements and cardiac markers in acute coronary syndromes. J. Trace Elem. Med. Biol. 18, 235-242.
  4. Bar-Or, D., Rael, L.T., Lau, E.P., Rao, N.K., Thomas, G.W., Winkler, J.V., Yukl, R.L., Kingston, R.G., and Curtis, C.G. (2001). An analog of the human albumin N-terminus (Asp-Ala-His-Lys) prevents formation of copper-induced reactive oxygen species. Biochem. Biophys. Res. Commun. 284, 856-862.
  5. Barbusinski, K. (2009). Fenton reaction-controversy concerning the chemistry. Ecol. Chem. Eng. S 16, 347-358.
  6. Barth, S., Glick, D., and Macleod, K.F. (2010). Autophagy: assays and artifacts. J. Pathol. 221, 117-124.
  7. Chevion, M., Jiang, Y., Har-El, R., Berenshtein, E., Uretzky, G., and Kitrossky, N. (1993). Copper and iron are mobilized following myocardial ischemia: possible predictive criteria for tissue injury. Proc. Natl. Acad. Sci. USA 90, 1102-1106.
  8. Choi, Y.J., Park, Y.J., Park, J.Y., Jeong, H.O., Kim, D.H., Ha, Y.M., Kim, J.M., Song, Y.M., Heo, H.S., Yu, B.P., et al. (2012). Inhibitory effect of mTOR activator MHY1485 on autophagy: suppression of lysosomal fusion. PLoS One 7, e43418.
  9. Clarke, M., Bennett, M., and Littlewood, T. (2007). Cell death in the cardiovascular system. Heart 93, 659-664.
  10. Diaz-Troya, S., Perez-Perez, M.E., Florencio, F.J., and Crespo, J.L. (2008). The role of TOR in autophagy regulation from yeast to plants and mammals. Autophagy 4, 851-865.
  11. Dimmeler, S., and Zeiher, A.M. (2000). Endothelial cell apoptosis in angiogenesis and vessel regression. Circ. Res. 87, 434-439.
  12. Dortwegt, R., and Maughan, E. (2001). The chemistry of copper in water and related studies planned at the advanced photon source. Conf. Proc. C0106181, 1456-1458
  13. Gallagher, C.H., and Reeve, V.E. (1971). Copper deficiency in the rat. Effect on liver and brain lipids. Aust. J. Exp. Biol. Med. Sci. 49, 453-461.
  14. Glick, D., Barth, S., and Macleod, K.F. (2010). Autophagy: cellular and molecular mechanisms. J. Pathol. 221, 3-12.
  15. He, C., Bassik, M.C., Moresi, V., Sun, K., Wei, Y., Zou, Z., An, Z., Loh, J., Fisher, J., Sun, Q., et al. (2012). Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis. Nature 481, 511-515.
  16. Hordyjewska, A., Popiolek, L., and Kocot, J. (2014). The many "faces" of copper in medicine and treatment. Biometals 27, 611-621.
  17. Ishida, S., Andreux, P., Poitry-Yamate, C., Auwerx, J., and Hanahan, D. (2013). Bioavailable copper modulates oxidative phosphorylation and growth of tumors. Proc. Natl. Acad. Sci. USA. 110, 19507-19512.
  18. Kim, J., Park, J., Choi, S., Chi, S.G., Mowbray, A.L., Jo, H., and Park, H. (2008). X-linked inhibitor of apoptosis protein is an important regulator of vascular endothelial growth factor-dependent bovine aortic endothelial cell survival. Circ. Res. 102, 896-904.
  19. Kundu, M., Lindsten, T., Yang, C.Y., Wu, J., Zhao, F., Zhang, J., Selak, M.A., Ney, P.A., and Thompson, C.B. (2008). Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation. Blood 112, 1493-1502.
  20. Laha, D., Pramanik, A., Maity, J., Mukherjee, A., Pramanik, P., Laskar, A., and Karmakar, P. (2014). Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7. Biochim. Biophys. Acta 1840, 1-9.
  21. Lee, H.R., Kim, J., Park, J., Ahn, S., Jeong, E., and Park, H. (2013). FERM domain promotes resveratrol-induced apoptosis in endothelial cells via inhibition of NO production. Biochem. Biophys. Res. Commun. 441, 891-896.
  22. Linder, M.C., and Hazegh-Azam, M. (1996). Copper biochemistry and molecular biology. Am. J. Clin. Nutr. 63, 797S-811S.
  23. Martinet, W., and De Meyer, G.R. (2009). Autophagy in atherosclerosis: a cell survival and death phenomenon with therapeutic potential. Circ. Res. 104, 304-317.
  24. Palmer, D.A., Benezeth, P., and Simonson, J.M. (2004). Solubility of copper oxides in water and steam. In 14th International Conference on the Properties of Water and Steam in Kyoto pp. 491-496.
  25. Powell, S.R., Gurzenda, E.M., Wingertzahn, M.A., and Wapnir, R.A. (1999). Promotion of copper excretion from the isolated rat heart attenuates postischemic cardiac oxidative injury. Am. J. Physiol-Heart C. 277, H956-H962.
  26. Rael, L.T., Rao, N.K., Thomas, G.W., Bar-Or, R., Curtis, C.G., and Bar-Or, D. (2007). Combined cupric- and cuprous-binding peptides are effective in preventing IL-8 release from endothelial cells and redox reactions. Biochem. Biophys. Res. Commun. 357, 543-548.
  27. Rajendran, R., Ren, M., Ning, P., Huat, B. T. K., Halliwell, B., and Watt, F. (2007). Promotion of atherogenesis by copper or iron-Which is more likely? Biochem. Biophys. Res. Commun. 353, 6-10.
  28. Rigo, A., Stevanato, R., Finazzi-Agro, A., and Rotilio, G. (1977). An attempt to evaluate the rate of the Haber-Weiss reaction by using OH radical scavengers. FEBS Lett. 80, 130-132.
  29. Robaye, B., Mosselmans, R., Fiers, W., Dumont, J.E., and Galand, P. (1991). Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro. Am. J. Pathol. 138, 447-453.
  30. Ryter, S.W., Lee, S.J., Smith, A., and Choi, A.M. (2010). Autophagy in vascular disease. Proc. Am. Thorac. Soc. 7, 40-47.
  31. Shaw, R.J. (2009). LKB1 and AMP-activated protein kinase control of mTOR signalling and growth. Acta Physiol. 196, 65-80.
  32. Shimomura, H., Terasaki, F., Hayashi, T., Kitaura, Y., Isomura, T., and Suma, H. (2001). Autophagic degeneration as a possible mechanism of myocardial cell death in dilated cardiomyopathy. Jpn. Circ. J. 65, 965-968.
  33. Shokrzadeh, M., Ghaemian, A., Salehifar, E., Aliakbari, S., Saravi, S.S., and Ebrahimi, P. (2009). Serum zinc and copper levels in ischemic cardiomyopathy. Biol. Trace Elem. Res. 127,116-123.
  34. Singh, I., Sagare, A.P., Coma, M., Perlmutter, D., Gelein, R., Bell, R.D., Deane, R.J., Zhong, E., Parisi, M., Ciszewski, J., et al. (2013). Low levels of copper disrupt brain amyloid-$\beta$ homeostasis by altering its production and clearance. Proc. Natl. Acad. Sci. USA 110, 14771-14776.
  35. Sun, T., Yan, Y., Zhao, Y., Guo, F., and Jiang, C. (2012). Copper oxide nanoparticles induce autophagic cell death in A549 cells. PLoS One 7, e43442.
  36. Szabo, C., Ischiropoulos, H., and Radi, R. (2007). Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat. Rev. Drug Discov. 6, 662-680.
  37. Tanaka, Y., Guhde, G., Suter, A., Eskelinen, E.L., Hartmann, D., Lullmann-Rauch, R., Janssen, P.M., Blanz, J., von Figura, K., and Saftig, P. (2000). Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 406, 902-906.
  38. Yu, K.N., Yoon, T.J., Minai-Tehrani, A., Kim, J.E., Park, S.J., Jeong, M.S., Ha, S.W., Lee, J.K., Kim, J.S., and Cho, M.H. (2013). Zinc oxide nanoparticle induced autophagic cell death and mitochondrial damage via reactive oxygen species generation. Toxicol. In Vitro 27, 1187-1195.
  39. Zhao, J.G., Yang, S.H., and Yang, S.G. (2012). Controllable onestep synthesis of CuO, Cu2O and Cu. Cryst. Res.Technol. 47, 1064-1068.

Cited by

  1. The double faced role of copper in Aβ homeostasis: A survey on the interrelationship between metal dyshomeostasis, UPS functioning and autophagy in neurodegeneration vol.347, 2017,
  2. Delphinidin-rich extracts of Hibiscus sabdariffa L. trigger mitochondria-derived autophagy and necrosis through reactive oxygen species in human breast cancer cells vol.25, 2016,
  3. Safe-by-Design CuO Nanoparticles via Fe-Doping, Cu–O Bond Length Variation, and Biological Assessment in Cells and Zebrafish Embryos vol.11, pp.1, 2017,
  4. Copper signalling: causes and consequences vol.16, pp.1, 2018,
  5. The protective role of autophagy in nephrotoxicity induced by bismuth nanoparticles through AMPK/mTOR pathway vol.12, pp.6, 2018,