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Copper-1,10-Phenanthroline-Induced Apoptosis in Liver Carcinoma Bel-7402 Cells Associates with Copper Overload, Reactive Oxygen Species Production, Glutathione Depletion and Oxidative DNA Damage

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Abstract

The mechanism of cytotoxicity on liver carcinoma Bel-7402 cells induced by copper-1,10-phenanthroline, Cu(OP)2, has been studied. Cell viability and apoptotic rate were examined in cells treated with Cu(OP)2 or Cu2+ alone. It was found that the apoptosis induced by Cu(OP)2 could not be induced by Cu2+ or OP alone in our experimental conditions. Total copper content in cells was measured by atomic absorption spectrophotometry, and the abnormal elevation of intracellular copper transported by lipophilic OP ligand may play the role of initial factor in the apoptosis, which caused subsequent redox state changes in cells. Intracellular levels of reactive oxygen species (ROS) were detected by fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Reduced (GSH) and total glutathione (GSSG + GSH) were determined by High-performance liquid chromatography (HPLC) after derivatization, and the ratios of GSH/GSSG were subsequently calculated. The overproduction of ROS and the decreased GSH/GSSG ratio were observed in cells which represented the occurrence of oxidative stress in the apoptosis. Oxidative DNA damage was also found in cells treated with Cu(OP)2 in the early stage of the apoptosis, and it suggests that the activation of DNA repair system may be involved in the pathway of the apoptosis induced by Cu(OP)2.

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References

  • Barbara SB, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313–20316

    Article  Google Scholar 

  • Bradford S (1976) Protein determination. Anal Biochem 72:248–252

    Article  PubMed  CAS  Google Scholar 

  • Burkitt MJ, Milne L, Nicotera P, Orrenius S (1996) 1,10-Phenanthroline stimulates internucleosomal DNA fragmentation in isolated rat-liver nuclei by promoting the redox activity of endogenous copper ions. Biochem J 313:163–170

    PubMed  CAS  Google Scholar 

  • Buttke TM, Sandstrom PA (1994) Oxidative stress as a mediator of apoptosis. Immunol Today 15:7–10

    Article  PubMed  CAS  Google Scholar 

  • Cereser C, Guichard J, Drai J (2001) Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts. J Chromatogr B 752:123–132

    Article  CAS  Google Scholar 

  • Chen F, Shi X (2002) Intracellular signal transduction of cells in response to carcinogenic metals. Crit Rev Oncol Hematol 42:105–121

    Article  PubMed  Google Scholar 

  • Collins AR (2004) The comet assay for DNA damage and repair. Mol Biotechnol 26:249–261

    Article  PubMed  CAS  Google Scholar 

  • Cotgreave IA, Gerdes RG (1998) Recent trends in glutathione biochemistry—glutathione–protein interactions: a molecular link between oxidative stress and cell proliferation? Biochem Biophys Res Commun 242:1–9

    Article  PubMed  CAS  Google Scholar 

  • Curtin JF, Donovan M, Cotter TG (2002) Regulation and measurement of oxidative stress in apoptosis. J Immunol Methods 265:49–72

    Article  PubMed  CAS  Google Scholar 

  • Danks DM (1988) Copper deficiency in humans. Annu Rev Nutr 8:235–257

    Article  PubMed  CAS  Google Scholar 

  • Dean RT, Fu S, Stocker R, Davies MJ (1997) Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 324:1–18

    PubMed  CAS  Google Scholar 

  • Dizdaroglu M, Aruoma OI, Halliwell B (1990) Modification of bases in DNA by copper ion-1,10-phenanthroline complexes. Biochemistry 29:8447–8451

    Article  PubMed  CAS  Google Scholar 

  • Gaetke LM, Chow CK (2003) Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology 189:147–163

    Article  PubMed  CAS  Google Scholar 

  • Galaris D, Evangelou A (2002) The role of oxidative stress in mechanisms of metal-induced carcinogenesis. Crit Rev Oncol Hematol 42:93–103

    Article  PubMed  Google Scholar 

  • Gerschenson LE, Rotello RL (1992) Apoptosis: A different type of cell death. FASEB J 6:2450–2455

    PubMed  CAS  Google Scholar 

  • Halliwell B, Aruoma OI (1991) DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian system. FEBS Lett 281:9–19

    Article  PubMed  CAS  Google Scholar 

  • Jabs T (1999) Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem Pharmacol 57:231–245

    Article  PubMed  CAS  Google Scholar 

  • Klatt P, Lamas S (2000) Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur J Biochem 267:4928–4944

    Article  PubMed  CAS  Google Scholar 

  • Krzysztof K, Anna L, Anna B, et al. (2003) A cross-platform public domain PC image-analysis program for the comet assay. Mut Res 534:15–20

    Google Scholar 

  • Linder MC (2001) Copper and genomic stability in mammals. Mut Res 475:141–152

    CAS  Google Scholar 

  • Ma Y, Cao L, Kawabata T, Yoshino T, Yang BB, Okada S (1998) Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells. Free Radic Biol Med 25:568–575

    Article  PubMed  CAS  Google Scholar 

  • Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  PubMed  CAS  Google Scholar 

  • Norbury CJ, Zhivotovsky B (2004) DNA damage-induced apoptosis. Oncogene 23:2797–2808

    Article  PubMed  CAS  Google Scholar 

  • Pourahmad J, Ross S, O‘Brien PJ (2001) Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+. Free Radic Biol Med 30:89–97

    Article  PubMed  CAS  Google Scholar 

  • Pulg S, Thiele DJ (2002) Molecular mechanism of copper uptake and distribution. Curr Opin Chem Biol 6:171–180

    Article  Google Scholar 

  • Ravia JJ, Stephen RM, Ghishan FK, Collins JF (2005) Menkes copper ATPase (Atp7a) is a novel metal-responsive gene in rat duodenum, and immunoreactive protein is present on brush-border and basolateral membrane domains. J Biol Chem 280:36221–36227

    Article  PubMed  CAS  Google Scholar 

  • Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30:1191–1212

    Article  PubMed  CAS  Google Scholar 

  • Schulz JB, Lindenau J, Seyfried J, Dichgans J (2000) Glutathione, oxidative stress and neurodegeneration. Eur J Biochem 267:4904–4911

    Article  PubMed  CAS  Google Scholar 

  • Sigman DS, Graham DR, D’Aurora V, Stern AM (1979) Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline cuprous complex. Inhibition of Escherichia coli DNA polymerase I. J Biol Chem 254:12269–12271

    PubMed  CAS  Google Scholar 

  • Singh RP, Kumar S, Nada R, Prasad R (2006) Evaluation of copper toxicity in isolated human peripheral blood mononuclear cells and it‘s attenuation by zinc: ex vivo. Mol Cell Biochem 282:13–21

    Article  PubMed  CAS  Google Scholar 

  • Sun Y, Oberley LW (1996) Redox regulation of transcriptional activators. Free Radic Biol Med 21:335–348

    Article  PubMed  CAS  Google Scholar 

  • Theophanides T, Anastassopoulou J (2002) Copper and carcinogenesis. Crit Rev Oncol Hematol 42:57–64

    Article  PubMed  CAS  Google Scholar 

  • Toyokuni S, Sagripanti JL (1996) Association between 8-hydroxy-2′-deoxyguanosine formation and DNA strand breaks mediated by copper and iron. Free Radic Biol Med 20:859–864

    Article  PubMed  CAS  Google Scholar 

  • Young MC, Yun SB, Soo YL (2003) Molecular ordering of ROS production, mitochondrial changes, and caspase activation during sodium salicylate-induced apoptosis. Free Radic Biol Med 34:434–442

    Article  Google Scholar 

  • Zhou H, Zheng C, Zou G, Tao D, Gong J (2002) G1-phase specific apoptosis in liver carcinoma cell line induced by copper-1,10-phenanthroline. Int J Biochem Cell Biol 34:678–684

    Article  PubMed  CAS  Google Scholar 

  • Zwart LL, Meerman JHN, Commandeur JNM, Vermeulen NPE (1999) Biomarkers of free radical damage. Applications in experimental animals and in humans. Free Radic Biol Med 26:202–226

    Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the grant from National Natural Science Foundation of China (Grant No. 30370366, 30470111) and The Research Fund for the Doctoral Program of Higher Education (RFDP).

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  1. State Key Laboratory of Virology, Department of Biotechnology, College of Life Sciences, Wuhan University, Wuhan, 430072, China

    Xiaoqiang Cai, Nina Pan & Guolin Zou

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  1. Xiaoqiang Cai
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  2. Nina Pan
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  3. Guolin Zou
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Correspondence to Guolin Zou.

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Cai, X., Pan, N. & Zou, G. Copper-1,10-Phenanthroline-Induced Apoptosis in Liver Carcinoma Bel-7402 Cells Associates with Copper Overload, Reactive Oxygen Species Production, Glutathione Depletion and Oxidative DNA Damage. Biometals 20, 1–11 (2007). https://doi.org/10.1007/s10534-006-9008-0

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  • DOI: https://doi.org/10.1007/s10534-006-9008-0

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