CuO nanoparticles induce cytotoxicity and apoptosis in human K562 cancer cell line via mitochondrial pathway, through reactive oxygen species and P53

Document Type : Original Article


1 Department of Biology and Institute of Biotechnology, Faculty of Sciences, Urmia University, Urmia, Iran

2 Microbiology Department, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran


Objective(s): This study focused on determining cytotoxic effects of copper oxide nanoparticles (CuO NPs) on chronic myeloid leukemia (CML) K562 cell line in a cell-specific manner and its possible mechanism of cell death. We investigated the cytotoxicity of CuO NPs against K562 cell line (cancerous cell) and peripheral blood mononuclear cell (normal cell).
Materials and Methods: The toxicity was evaluated using cell viability, oxidative stress and apoptosis detection. In addition, the expression levels of P53, Caspase 3, Bcl-2, and Bax genes in K562 cells were studied by reverse transcription polymerase chain reaction (RT-PCR) analysis.
Results: CuO NPs exerted distinct effects on cell viability via selective killing of cancer cells in a dose-dependent manner while not impacting normal cells in MTT assay. The dose-dependent cytotoxicity of CuO NPs against K562 cells was shown through reactive oxygen species (ROS) generation. The CuO NPs induced apoptosis was confirmed through acridine orange and propidium iodide double staining. Tumor suppressor gene P53 was up regulated due to CuO NPs exposure, and increase in Bax/Bcl-2 ratio suggested mitochondria-mediated pathway is involved in CuO NPs induced apoptosis. We also observed that Caspase 3 gene expression remained unchanged up to 24 hr exposure.                                                                            
Conclusion: These molecular alterations provide an insight into CuO NPs-caused inhibition of growth, generation of ROS, and apoptotic death of K562 cells.


1.Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000; 96:3343-3356.
2.Druker BJ, Guilhot F, O'Brien SG, Gathmann I, Kantarjian H, Gattermann N, et al. Five year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 2006; 355:2408–2417.
3.Rasmussen JW, Martinez E, Louka P, Wingett DG. Zinc oxide nanoparticles for selective destruction of tumor cells and potential for drug delivery applications. Expert Opin Drug Deliv 2012; 7:1063–1077.
4.Manimaran R, Palaniradja K, Alagumurthi N, Sendhilnathan S, Hussain J. Preparation and characterization of copper oxide nanofluid for heat transfer applications. Appl Nanosci 2014; 4:163–167.
5.Chang YN, Zhang M, Xia L, Zhang J, Xing L. The toxic effects and mechanisms of CuO and ZnO nanoparticles. Materials 2012; 5:2850-2871.
6.Yang H, Liu C, Yang DF, Zhang HS, Xi Z. Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: The role of particle size, shape and composition. J Appl Toxicol 2009; 29:69–78.
7.Ott M, Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria, oxidative stress and cell death. Apoptosis 2007; 12:913–922.
8.Li T, Kon N, Jiang L, Tan M, Ludwig T, Zhao Y, et al. Tumor suppression in the absence of P53-mediated cell-cycle arrest, apoptosis, and senescence. Cell 2012; 149:1269–1283.
9.Antonsson B, Martinou JC. TheBcl-2 protein family. Exp Cell Res 2000; 256:50–57.
10.Chougule M, Patel AR, Sachdeva P, Jackson T, Singh M. Anticancer activity of Noscapine, an opioid alkaloid in combination with cisplatin in human non-small cell lung cancer. Lung Cancer 2011; 71:271–282.
11.Green DR, Reed JC. Mitochondria and apoptosis. Science1998; 281:1309–1312.
12.Boyum A. Separation of leukocytes from blood and bone marrow. Introduction. Scand J Clin Lab Invest Suppl 1968; 97:7-11.
13.Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.  J Immunol Methods 1983; 65:55-63.
14.Elbekai RH, El-Kadi AOS. The role of oxidative stress in the modulation of aryl hydrocarbon receptor-regulated genes by As3+, Cd2+ and Cr6+. Free Radic Biol Med 2005; 39:1499–1511.
15.Ali R, Alabs AM, Ali AM, Ideris A, Omar AR, Yusoff K, et al. Cytolytic effects and apoptosis induction of new castle disease virus strain AF2240 on anaplastic astrocytoma brain tumor cell line. Neurochem Res 2011; 36:2051-2062.
16.Wu YN, Yang LX, Shi XY, Li IC, Biazik JM, Ratinac KR, et al. The selective growth inhibition of               oral cancer by iron core-gold shell nanoparticles through mitochondria-mediated autophagy. Bio-materials 2011; 32:4565–4573.
17.Premanathan M, Karthikeyan K, Jeyasubramanian K, Manivannan G. Selective toxicity of ZnO nanoparticles toward Gram positive bacteria and cancer cells by apoptosis through lipid peroxidation. Nanomedicine 2011; 7:184–192.
18.Ahamed M, Alhadlaq HA, Khan MAM, Akhtar MJ.  Selective killing of cancer cells by iron oxide nanoparticles mediated through reactive oxygen species via P53 pathway. J Nanopart Res 2013; 15:1225.
19.Ryter SW, Kim HP, Hoetzel A, Park JW, NakahiraK, Wang X, et al. Mechanisms of cell death in oxidative stress. Antioxid Redox Signal 2007; 9:49–89.
20.Ahamed M, Siddiqui MA, Akhtar MJ, Ahmad I, Pant AB, Alhadlaq HA. Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells. Biochem Biophys Res Commun 2010; 396 578–583.
21.Sun J, Wang S, Zhao D, Hun FH, Weng L, Liu H. Cytotoxicity, permeability, and inflammation of metal oxide nanoparticles in human cardiac micro-vascular endothelial cells: cytotoxicity, permeability, and inflammation of metal oxide nanoparticles. Cell Biol Toxicol 2011; 27:333–342.
22.Siddiqui MA, Alhadlaq HA, Ahmad J, Al-Khedhairy AA, Musarrat J, Ahamed M. Copper oxide nanoparticles induced mitochondria mediated apoptosis in human hepato carcinoma cells. PLoS One 2013; 8:e69534.
23.Perreault F, Melegari SP, Costa CH, Siddi-Rossetto AF, Popovic R, Matias WG. Genotoxic effects of copper oxide nanoparticles in Neuro2A cell cultures. Sci Total Environ 2012; 441: 117–124.
24.Doonan F, Cotter TG. Morphological assessment of apoptosis. Methods 2008; 44:200-204.                         
25.Chowdhury R, Chowdhury S, Roychoudhury P, Mandal C,Chaudhuri K. Arsenic induced apoptosis in malignant melanoma cells is enhanced by menadione through ROS generation, p38 signaling and P53 activation. Apoptosis 2009; 14:108–123.
26.Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol in vitro 2005; 19:975–983.
27.Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebemann DA, et al. Tumor suppressor P53 is regulator of bcl-2 and bax gene expression  in vitro and in vivo. Oncogene 1994; 9:1799.
28.Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998; 281:1322-1326.
29.Zhang GJ, Kimijima I, Onda M, Kanno M, Sato H, Watanabe T, et al. Tamoxifen-induced apoptosis in breast cancer cells relates to down regulation of Bcl-2, but not Bax and Bcl-xL, without alteration of P53 protein levels. Clin Cancer Res 1999; 5:2971-2977.
30.Chen M, Quintans J, Fuks Z, Thompson C, Kufe DW, Weichselbaum RR. Suppression of Bcl-2 messenger RNA production may mediate apoptosis after ionizing radiation, tumor necrosis factor a, and ceramide. Cancer Res 1995; 55:991–994.
31.Mroz RM, Schins RP, Li H, Jimenez LA, Drost    EM, Holownia A, et al. Nanoparticle driven DNA damage mimics irradiation-related carcinogenesis pathways. Eur Respir J 2008; 31:241– 251.
32.Yang SH, Lu MC, Chien CM, Tsai CH, Lu YJ, Hour TC, et al.  Induction of apoptosis in human leukemia K562 cells by cardiotoxin III. Life Sci 2005; 76:2513–2522.
33.Sánchez-Pérez Y, Chirino YI, Osornio-Vargas AR, Morales-Bárcenas R, Gutiérrez-Ruíz C, Vázquez-LópezI, et al. DNA damage response of A549 cells treated with particulate matter (PM10) of urban air pollutants Cancer Lett 2009; 278:192–200.
34.Yang SH , Tsai CH, Lu MC , Yang  YN, Chien CM, Lin SF, et al. Effects of cardiotoxin III on expression of genes and proteins related to G2/M arrest and apoptosis in K562 cells. Mol Cell Biochem 2007; 300:185–190.