Tumor Biology

, Volume 36, Issue 5, pp 3661–3668 | Cite as

Calcitriol induced redox imbalance and DNA breakage in cells sharing a common metabolic feature of malignancies: Interaction with cellular copper (II) ions leads to the production of reactive oxygen species

Research Article


Calcitriol is known to selectively kill malignant cells, however, not much is known about the mechanism by which it kills malignant cells and spares the “normal” cells. Since elevation of cellular copper is a metabolic condition common to all malignancies, we developed a mouse model to mimic this condition and treated the animals with calcitriol. It was observed that calcitriol–copper interaction in vivo causes severe fluctuations in cellular enzymatic and nonenzymatic scavengers of reactive oxygen species (ROS). Lipid peroxidation, a well-established marker of oxidative stress, was found to increase, and a substantial cellular DNA breakage was observed. Calcitriol–copper interaction in vivo was observed to lead the cells to an apoptosis like cell death. We propose that the interaction of calcitriol and copper within malignant cells and the consequent redox scavenger fluctuations and ROS-mediated DNA breakage may be one of the several mechanisms by which calcitriol causes selective cell death of malignant cells, while sparing normal cells.


Cancer Calcitriol Copper Cell death ROS 



Analysis of variance


Reactive oxygen species


Standard error of mean


Thiobarbituric acid reactive substances


  1. 1.
    Yoshida D, Ikada Y, Nakayama S. Quantitative analysis of copper, zinc and copper/zinc ratio in selective human brain tumors. J Neuro Oncol. 1993;16:109–15.CrossRefGoogle Scholar
  2. 2.
    Ebad E, Swanson S. The status of zinc, copper and metallothionein in cancer patients. Prog Clin Biol Res. 1998;259:167–75.Google Scholar
  3. 3.
    Nasulewis A, Mazur A, Opolski A. Role of copper in angiogenesis: clinical-implication. J Trace Elem Med Biol. 2004;18:1–8.CrossRefGoogle Scholar
  4. 4.
    Zowczak M, Iskra M, Ski LT, Cofta S. Analysis of serum copper and zinc concentrations in cancer patients. Biol Trace Elem Res. 2001;82:8201–3.CrossRefGoogle Scholar
  5. 5.
    Kagawa TF, Geierstanger BH, Wang AJH, Ho PS. Covalent modification of guanine bases in double stranded DNA: the 1:2-AZ-DNA structure of dc(CACACG) in the presence of CuCl2. J Biol Chem. 1991;266:20175–84.PubMedGoogle Scholar
  6. 6.
    Christakos S, Ajibade DV, Dhawan P, Fechner AJ, Mady LJ. Vitamin D: metabolism. Endo Metab Clin North Am. 2010;39:243–53.CrossRefGoogle Scholar
  7. 7.
    Studzinski GP, Moore DC. Sunlight—can it prevent as well as cause cancer? Cancer Res. 1995;55:4014–22.PubMedGoogle Scholar
  8. 8.
    Moran PO, Larriba MJ, Franco NP, Aguilera O, Sancho JMG, Munzo A. Vitamin D and cancer: an update on in vitro and in vivo data. Fron Bio. 2005;10:2723–49.CrossRefGoogle Scholar
  9. 9.
    Garland CF, Garland FC, Gorham ED, Lipkin M, Newmark H, Mohr SB, et al. Role of vitamin D in cancer prevention. Am J Pub Health. 2006;96:252–61.CrossRefGoogle Scholar
  10. 10.
    Garland CF, Gorham ED, Mohr SB, Garland FB. Vitamin D for cancer prevention: global perspective. Ann Epidemiol. 2009;19:468–83.CrossRefPubMedGoogle Scholar
  11. 11.
    Rizvi A, Chibber S, Naseem I. Cu(II)-vitamin D interaction leads to free radical-mediated cellular DNA damage: a novel putative mechanism for its selective cytotoxic action against malignant cells. Tumour Biol. 2014. doi: 10.1007/s13277-014-2770-7.Google Scholar
  12. 12.
    Rizvi A, Hasan SS, Naseem I. Selective cytotoxic action and DNA damage by calcitriol–Cu(II) interaction: putative mechanism of cancer prevention. PLoS One. 2013. doi: 10.1371/journal.pone.0076191.PubMedPubMedCentralGoogle Scholar
  13. 13.
    Hasan SS, Rizvi A, Naseem I. Calcitriol induced DNA damage: towards a molecular mechanism of selective cell death. IUBMB Life. 2013. doi: 10.1002/iub.1199.PubMedGoogle Scholar
  14. 14.
    Spector T. Refinement of Coomassie blue method of protein quantitation. Anal Biochem. 1978;86:42–146.CrossRefGoogle Scholar
  15. 15.
    Beuge JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302–10.CrossRefGoogle Scholar
  16. 16.
    Marklund S, Marklund G. The involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47:469–74.CrossRefPubMedGoogle Scholar
  17. 17.
    Abei H. Catalse in vitro. Methods Enzymol. 1984;105:121–6.CrossRefGoogle Scholar
  18. 18.
    Habig W, Pabst MJ, Jacoby WH. Glutathione-S-transferases: the first step in mercapturic acid formation. J Biol Chem. 1974;249:7130–9.PubMedGoogle Scholar
  19. 19.
    Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR. Bromobenzene induced liver necrosis: protective role of glutathione and evidence for 3,4- bromobenzene oxide as the hepatotoxic metabolite. Pharmacology. 1974;11:151–69.CrossRefPubMedGoogle Scholar
  20. 20.
    Hassan I, Chibber S, Khan AA, Naseem I. Riboflavin ameliorates cisplatin induced toxicities under photoillumination. PLoS One. 2012;7(5):e36273. doi: 10.1371/journal.pone.0036273.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hadi SM, Asad SF, Singh S, Ahmad A. Putative mechanism for anticancer and apoptosis inducing properties of plant derived polyphenolic compounds. IUBMB Life. 2002;50:167–71. doi: 10.1080/152165400300001471.Google Scholar
  22. 22.
    Chibber S, Farhan M, Hassan I, Naseem I. White light mediated Cu(II)-5FU interaction, augments chemotherapeutic potential of 5FU: an in vitro study. Tumour Biol. 2011;32:881–92. doi: 10.1007/s13277-011-0189-y.CrossRefPubMedGoogle Scholar
  23. 23.
    Chibber S, Farhan M, Hassan I, Naseem I. Light mediated intraction of methotrexate with transition metal Cu(II). Med Chem Res. 2011. doi: 10.1007/s00044-011-9758-2.Google Scholar
  24. 24.
    Hadi SM, Bhat SH, Azmi AS, Hanif S, Shamim U, Ullah MF. Oxidative breakage of cellular DNA by plant polyphenols: putative mechanism for their anticancer properties. Semin Cancer Biol. 2007;17:370–6. doi: 10.1016/j.semcancer.2007.04.002. PubMed: 17572102.CrossRefPubMedGoogle Scholar
  25. 25.
    Ullah MF, Khan HY, Zubair H, Shamim U, Hadi SM. The antioxidant ascorbic acid mobilizes nuclear copper leading to prooxidant breakage of cellular DNA: implications for chemotherapeutic action against cancer. Chemoth and Pharamacol. 2011;67:103–10. doi: 10.1007/s00280-010-1290-4.CrossRefGoogle Scholar
  26. 26.
    Ramanathan R, Das NP, Tan CH. Effect of g-linolenic acid, flavanoids and vitamins on cytotoxicity and lipid peroxidation. Free Radic Biol Med. 1994;16:43–8.CrossRefPubMedGoogle Scholar
  27. 27.
    Koren R, Hadari-Naor I, Zuck E, Rotem C, Liberman UA, et al. Vitamin D is a prooxidant in breast cancer. Cancer Res. 2001;61:1439–44.PubMedGoogle Scholar
  28. 28.
    Narvaez CJ, Welsh J. Role of mitochondria and caspases in Vit D mediated cell death of MCF 7 breast cancer cells. JBC. 2001;276:9101–7.CrossRefGoogle Scholar
  29. 29.
    Ravid A, Rocker D, Machlenkin A, Rotem C, Hochman A, Kessler-Icekson G, et al. 1,25 dihydroxy vitamin D3 enhances the suseptiblity of breast cancer cells to doxorubicin induced oxidative damage. Cancer Res. 1999;59:862–7.PubMedGoogle Scholar
  30. 30.
    Koren R, Rocker D, Kotestiano O, Liberman UA, Ravid A. Synergistic anticancer activity of 1,25 dihydroxy vitamin D3 and immune cytokines: involvement of reactive oxygen species. J Steroid Biochem Mol Biol. 2000;73:105–12.CrossRefPubMedGoogle Scholar
  31. 31.
    Liochev SL. The role of iron sulphur cluster in vivo hydroxyl radical production. Free Rad Res. 1996;25:369–84.CrossRefGoogle Scholar
  32. 32.
    Koren R, Ravid A. Vitamin D and cellular response to oxidative stress. In: Feildman D, editor. Vitamin D. 2nd ed. Boston: Elsevier Academic Press; 2005. p. 761–73.CrossRefGoogle Scholar
  33. 33.
    Weitsman GE, Koren R, Zuck E, Rotem C, Liberman UA, Ravid A. Vitamin D sensitizes breast cancer cells to the action of H2O2. Mitochondria as a convergence point in cell death pathway. Free Rad Biol Med. 2005;39:266–78.CrossRefPubMedGoogle Scholar
  34. 34.
    Polla BS, Bonventre JV, Krane SM. 1,25 dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in human monocytic line U937: role of calcium and heat shock. J Cell Biol. 1988;107:373–80.CrossRefPubMedGoogle Scholar
  35. 35.
    Diker Cohen T, Koren R, Libermann UA, Ravid A. Vitamin D protects keratinocytes from apoptosis induced by osmotc shock, oxidative stress, and tumour necrosis factor. Ann NY Acad Sci. 2003;1010:350–3.CrossRefPubMedGoogle Scholar
  36. 36.
    Leist M, Jaattela M. Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol. 2001;2:589–98.CrossRefPubMedGoogle Scholar
  37. 37.
    Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resist Updates. 2004;7:97–110.CrossRefGoogle Scholar
  38. 38.
    Zhou Y, Hileman EO, Plunkett W, Keating MJ, Huang P. Free radical stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents. Blood. 2003;101:4098–104.CrossRefPubMedGoogle Scholar
  39. 39.
    Geller HM, Cheng KY, Goldsmith NK, Romero AA, Zhang AL, Morris EJ, et al. Oxidative stress mediates neuronal DNA damage and apoptosis in response to cytosine arabinoside. J Neurochem. 2001;78:265–75.CrossRefPubMedGoogle Scholar
  40. 40.
    Ramanathan B, Jan KY, Chen CH, Hour TC, Yu HJ, Pu YS. Resistance to paclitaxel is proportional to cellular total antioxidant capacity. Cancer Res. 2005;65:8455–60.CrossRefPubMedGoogle Scholar
  41. 41.
    Weydert CJ, Waugh TA, Ritchie JM, Iyer KS, Smith JL, Li L, et al. Overexpression of manganese or copper-zinc superoxide dismutase inhibits breast cancer growth. Free Radical Biol Med. 2006;41:226–37.CrossRefGoogle Scholar
  42. 42.
    Oberley LW. Mechanism of the tumor suppressive effect of MnSOD overexpression. Biomed Pharmacother. 2005;59:143–8.CrossRefPubMedGoogle Scholar
  43. 43.
    Wang M, Kirk JS, Venkataraman S, Domann FE, Zhang HJ, Schafer FQ, et al. Manganese superoxide dismutase suppresses hypoxic induction of hypoxia-inducible factor-1α and vascular endothelial growth factor. Oncogene. 2005;24:8154–66.PubMedGoogle Scholar
  44. 44.
    Kinnula VL, Crapo JD. Superoxide dismutases in malignant cells and human tumors. Free Radical Biol Med. 2004;36:718–44.CrossRefGoogle Scholar
  45. 45.
    Furuta J, Nobeyama Y, Umebayashi Y, Otsuka F, Kikuchi K, Ushijima T. Silencing of peroxiredoxin 2 and aberrant methylation of 33 CpG islands in putative promoter regions in human malignant melanomas. Cancer Res. 2006;66:6080–6.CrossRefPubMedGoogle Scholar
  46. 46.
    Mangelsdorf DJ, Thummel CB, Herrlich M, Schütz P, Umesono G, Blumberg K, et al. The nuclear receptor superfamily: the second decade. Cell. 1995;83:835–9.CrossRefPubMedGoogle Scholar
  47. 47.
    Freedman LP. Transcriptional targets of the vitamin D3 receptor-mediating cell cycle arrest and differentiation. J Nutr. 1999;129:581s–6s.PubMedGoogle Scholar
  48. 48.
    Deeb KK, Trump DL, Johnson CS. Vitamin D signalling pathways in cancer: potential for anticancer therapeutics. Nat Rev Can. 2007;7:684–700.CrossRefGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  1. 1.Department of Biochemistry, Faculty of Life SciencesAligarh Muslim UniversityAligarhIndia
  2. 2.Section of Radiobiology, Department of Radiology, The Biomedical Research TowerThe Ohio State UniversityColumbusUSA
  3. 3.Department of PathologyGovernment Medical CollegeHaldwaniIndia

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