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CYP1A1 activation of aminoflavone leads to DNA damage in human tumor cell lines

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Abstract

Purpose: Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one; AF; NSC 686288) is a novel anticancer agent with a unique pattern of growth inhibitory activity in the National Cancer Institute (NCI) 60 tumor cell line screen. Phase I clinical trials with AF will begin soon. We previously demonstrated extensive metabolism of AF by cytochrome P450 (CYP) 1A1 and CYP1A2, metabolic activation, formation of irreversible protein and DNA adducts and p53 stabilization in sensitive, but not resistant, human tumor cell lines treated with AF [9]. The present studies focus on the effects of AF on cellular DNA and cellular responses to DNA damage. Methods: Phosphorylation of H2AX in MCF7 cells treated with AF was determined with immunofluorescence. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) assays were used to determine the effect of cotreatment with caffeine or wortmannin, inhibitors of ataxia-telangiectasia-mutated protein (ATM) and ATR (ATM- and rad3-related protein), on the AF IC50 values for MCF7 cells. DNA damage in MCF7 cells treated with AF was determined by alkaline elution. DNA-topoisomerase complex stabilization was ascertained by the ICE (in vitro complex of enzyme) assay. Results: Treatment of sensitive cells with AF resulted in phosphorylation of H2AX, a histone 2A variant that is phosphorylated in response to DNA damage. AF IC50 values for MCF7 cells were lowered by cotreatment with caffeine or wortmannin, further implicating DNA damage in AF cytotoxicity. There was no evidence of DNA–DNA cross-linking in sensitive cells, but protein-associated single-strand breaks were observed after AF treatment. Although this pattern of DNA damage is commonly associated with topoisomerase poisons, there was no evidence for AF-induced stabilization of either topoisomerase I- or II-DNA complexes. Conclusions: These studies further implicate DNA damage in the cytotoxicity of AF and identify biochemical features of that damage including formation of protein-associated single-strand breaks not involving topoisomerase I or II.

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References

  1. Abraham RT (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 15(17):2177–2196

    Article  PubMed  CAS  Google Scholar 

  2. Akama T, Shida Y, Sugaya T, Ishida H, Gomi K, Kasai M (1996) Novel 5-aminoflavone derivatives as specific antitumor agents in breast cancer. J Med Chem 39:3461–3469

    Article  PubMed  CAS  Google Scholar 

  3. Alley MC, Stinson SF, Pacula-Cox CM, Camalier RF, Phillips LR, Daw TW, Sausville EA (1999) Pharmacologic evaluations of a novel amino-substituted flavone (NSC 686288) exhibited unique in vitro and in vivo anticancer activities. Proc Am Assoc Cancer Res 40:119

    Google Scholar 

  4. Aune GJ, Furuta T, Pommier Y (2002) Ecteinascidin 743: a novel anticancer drug with a unique mechanism of action. Anticancer Drugs 13(6):545–55

    Article  PubMed  CAS  Google Scholar 

  5. Cushman M, Zhu H, Geahlen RL, Kraker AJ (1994) Synthesis and biochemical evaluation of a series of aminoflavones as potential inhibitors of protein-tyrosine kinases p561cd, EGF4, and p60v-src. J Med Chem 37:3353–3362

    Article  PubMed  CAS  Google Scholar 

  6. Ferriola PC, Cody V, Middleton E Jr (1989) Protein kinase C inhibition by plant flavonoids: kinetic mechanisms and structure-activity relationships. Biochem Pharmacol 38:1617–1624

    Article  PubMed  CAS  Google Scholar 

  7. Khan QA, Kohlhagen G, Marshall R, Austin CA, Kalena GP, Kroth H, Sayer JM, Jerina DM, Pommier Y (2003) Position-specific trapping of topoisomerase II by benzo[a]pyrene diol epoxide adducts: implications for interactions with intercalating anticancer agents. Proc Natl Acad Sci USA 100(21):12498–503

    Article  PubMed  CAS  Google Scholar 

  8. Kohn KW, Ewig RA, Erickson L, Zweeling L (1981) Measurement of strand breaks and crosslinks in DNA by alkaline elution. In: Friedberg EC, Hanawalt PC (eds) DNA Repair: a laboratory manual of research techniques. 379 p

  9. Kuffel MJ, Schroeder JC, Pobst LJ, Naylor S, Reid JM, Kaufmann SH, Ames MM (2002) Activation of the antitumor agent aminoflavone (NSC 686288) is mediated by induction of tumor cell cytochrome P450 1A1/1A2. Mol Pharmacol 62(1):143–153

    Article  PubMed  CAS  Google Scholar 

  10. Liu LF, Rowe TC, Yang L, Tewey KM, Chen GL (1983) Cleavage of DNA by mammalian DNA topoisomerase II. J Biol Chem 258(24):15365–15370

    PubMed  CAS  Google Scholar 

  11. Mao Y, Okada S, Chang LS, Muller MT (2000) p53 dependence of topoisomerase I recruitment in vivo. Cancer Res 60(16):4538–4543

    PubMed  CAS  Google Scholar 

  12. Nelson EM, Tewey KM, Liu LF (1984) Mechanism of antitumor drug action: poisoning of mammalian DNA topoisomerase II on DNA by 4′-(9-acridinylamino)-methanesulfon-m-anisidide. Proc Natl Acad Sci USA 81(5):1361–1365

    Article  PubMed  CAS  Google Scholar 

  13. Nielsen SE, Breinholt V, Justensen U, Cornett C, Dragsted LO (1998) In vitro biotransformation of flavonoids by rat liver microsomes. Xenobiotica 28(4):389–401

    Article  PubMed  CAS  Google Scholar 

  14. Pommier Y, Kohlhagen G, Laco GS, Kroth H, Sayer JM, Jerina DM (2002) Different effects on human topoisomerase I by minor groove and intercalated deoxyguanosine adducts derived from two polycyclic aromatic hydrocarbon diol epoxides at or near a normal cleavage site. J Biol Chem 277(16):13666–13672

    Article  PubMed  CAS  Google Scholar 

  15. Pommier Y, Schwartz RE, Zwelling LA, Kerrigan D, Mattern MR, Charcosset JY, Jacquemin-Sablon A, Kohn KW (1986) Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibitors. Cancer Res 46(2):611–616

    PubMed  CAS  Google Scholar 

  16. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273(10):5858–5868

    Article  PubMed  CAS  Google Scholar 

  17. Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Karnitz LM, Abraham RT (1999) Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 59(17):4375–4382

    PubMed  CAS  Google Scholar 

  18. Sarkaria JN, Tibbetts RS, Busby EC, Kennedy AP, Hill DE, Abraham RT (1998) Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res 58(19):4375–4382

    PubMed  CAS  Google Scholar 

  19. Shi Q, Chem K, Morris-Natschke SL, Lee KH (1998) Recent progress in the development of tubulin inhibitors as antimitotic antitumor agents. Curr Pharm Des 4:219–248

    PubMed  CAS  Google Scholar 

  20. Szmigiero L, Erickson LC, Ewig RA, Kohn KW (1984) DNA strand scission and cross-linking by diaziridinylbenzoquinone (diaziquone) in human cells and relation to cell killing. Cancer Res 44(10):4447–4452

    PubMed  CAS  Google Scholar 

  21. Takebayashi Y, Goldwasser F, Urasaki Y, Kohlhagen G, Pommier Y (2001) Ecteinascidin 743 induces protein-linked DNA breaks in human colon carcinoma HCT116 cells and is cytotoxic independently of topoisomerase I expression. Clin Cancer Res 7(1):185–191

    PubMed  CAS  Google Scholar 

  22. Takebayashi Y, Pourquier P, Zimonjic DB, Nakayama K, Emmert S, Ueda T, Urasaki Y, Kanzaki A, Akiyama SI, Popescu N, Kraemer KH, Pommier Y (2001) Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair. Nat Med 7(8):961–966

    Article  PubMed  CAS  Google Scholar 

  23. Trask DK, Muller MT (1988) Stabilization of type I topoisomerase-DNA covalent complexes by actinomycin D. Proc Natl Acad Sci USA 85(5):1417–1421

    Article  PubMed  CAS  Google Scholar 

  24. Ward IM, Chen J (2001) Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J Biol Chem 276(51):47759–47762

    Article  PubMed  CAS  Google Scholar 

  25. Weinstein JN, Myers TG, O’Connor PM, Friend SH, Fornace AJ Jr, Kohn KW, Fojo T, Bates SE, Rubinstein LV, Anderson NL, Buolamwini JK, van Osdol WW, Monks AP, Scudiero DA, Sausville EA, Zaharevitz DW, Bunow B, Viswanadhan VN, Johnson GS, Wittes RE, Paull KD (1997) An information-intensive approach to the molecular pharmacology of cancer. Science 275(5298):343–349

    Article  PubMed  CAS  Google Scholar 

  26. Yamashita Y, Kawada S, Nakano H (1990) Induction of mammalian topoisomerase II dependent DNA cleavage by nonintercalative flavonoids, genistein and orobol. Biochem Pharmacol 39:737–744

    Article  PubMed  CAS  Google Scholar 

  27. Zhai S, Dai R, Friedman FK, Vestal RE (1998) Comparative inhibition of human cytochromes P450 1A1 and 1A2 by flavonoids. Drug Metab Dispos 26(10):989–992

    PubMed  CAS  Google Scholar 

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Acknowledgements

The authors wish to thank Dr. Junjie Chen for generously providing the γ-H2AX antibody. This work was funded by a Mary Kay Ash Foundation Grant (MMA) and the Mayo Comprehensive Cancer Center (CA15083).

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  1. Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Guggenheim 13, 200 First Street S.W., Rochester, MN, 55905, USA

    Lori J. Pobst & Matthew M. Ames

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  1. Lori J. Pobst
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Correspondence to Matthew M. Ames.

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Pobst, L.J., Ames, M.M. CYP1A1 activation of aminoflavone leads to DNA damage in human tumor cell lines. Cancer Chemother Pharmacol 57, 569–576 (2006). https://doi.org/10.1007/s00280-005-0075-7

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