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Thioredoxin reductase is inhibited by the carbamoylating activity of the anticancer sulfonylhydrazine drug laromustine

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Abstract

The thioredoxin system facilitates proliferative processes in cells and is upregulated in many cancers. The activities of both thioredoxin (Trx) and its reductase (TrxR) are mediated by oxidation/reduction reactions among cysteine residues. A common target in preclinical anticancer research, TrxR is reported here to be significantly inhibited by the anticancer agent laromustine. This agent, which has been in clinical trials for acute myelogenous leukemia and glioblastoma multiforme, is understood to be cytotoxic principally via interstrand DNA crosslinking that originates from a 2-chloroethylating species generated upon activation in situ. The spontaneous decomposition of laromustine also yields methyl isocyanate, which readily carbamoylates thiols and primary amines. Purified rat liver TrxR was inhibited by laromustine with a clinically relevant IC50 value of 4.65 μM. A derivative of laromustine that lacks carbamoylating activity did not appreciably inhibit TrxR while another derivative, lacking only the 2-chloroethylating activity, retained its inhibitory potency. Furthermore, in assays measuring TrxR activity in murine cell lysates, a similar pattern of inhibition among these compounds was observed. These data contrast with previous studies demonstrating that glutathione reductase, another enzyme that relies on cysteine-mediated redox chemistry, was not inhibited by methylcarbamoylating agents when measured in cell lysates. Mass spectrometry of laromustine-treated enzyme revealed significant carbamoylation of TrxR, albeit not on known catalytically active residues. However, there was no evidence of 2-chloroethylation anywhere on the protein. The inhibition of TrxR is likely to contribute to the cytotoxic, anticancer mechanism of action for laromustine.

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References

  1. Baumann RP, Seow HA, Shyam K, Penketh PG, Sartorelli AC (2005) The antineoplastic efficacy of the prodrug cloretazine is produced by the synergistic interaction of carbamoylating and alkylating products of its activation. Oncol Res 15:313–325

    PubMed  CAS  Google Scholar 

  2. Finch RA, Shyam K, Penketh PG, Sartorelli AC (2001) 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101 M): a novel sulfonylhydrazine prodrug with broad-spectrum antineoplastic activity. Cancer Res 61:3033–3038

    PubMed  CAS  Google Scholar 

  3. Ishiguro K, Seow HA, Penketh PG, Shyam K, Sartorelli AC (2006) Mode of action of the chloroethylating and carbamoylating moieties of the prodrug cloretazine. Mol Cancer Ther 5:969–976

    Article  PubMed  CAS  Google Scholar 

  4. Penketh PG, Shyam K, Baumann RP, Remack JS, Brent TP, Sartorelli AC (2004) 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazi ne (VNP40101M): I. Direct inhibition of O6-alkylguanine-DNA alkyltransferase (AGT) by electrophilic species generated by decomposition. Cancer Chemother Pharmacol 53:279–287

    Article  PubMed  CAS  Google Scholar 

  5. Penketh PG, Baumann RP, Ishiguro K, Shyam K, Seow HA, Sartorelli AC (2008) Lethality to leukemia cell lines of DNA interstrand cross-links generated by cloretazine derived alkylating species. Leuk Res 32:1546–1553

    Article  PubMed  CAS  Google Scholar 

  6. Baumann RP, Shyam K, Penketh PG, Remack JS, Brent TP, Sartorelli AC (2004) 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazi ne (VNP40101M): II. Role of O6-alkylguanine-DNA alkyltransferase in cytotoxicity. Cancer Chemother Pharmacol 53:288–295

    Article  PubMed  CAS  Google Scholar 

  7. Frederick AM, Davis ML, Rice KP (2009) Inhibition of human DNA polymerase beta activity by the anticancer prodrug cloretazine. Biochem Biophys Res Commun 378:419–423

    Article  PubMed  CAS  Google Scholar 

  8. Eisenbrand G, Muller N, Denkel E, Sterzel W (1986) DNA adducts and DNA damage by antineoplastic and carcinogenic N-nitrosocompounds. J Cancer Res Clin Oncol 112:196–204

    Article  PubMed  CAS  Google Scholar 

  9. Gombar CT, Tong WP, Ludlum DB (1980) Mechanism of action of the nitrosoureas–IV. Reactions of bis-chloroethyl nitrosourea and chloroethyl cyclohexyl nitrosourea with deoxyribonucleic acid. Biochem Pharmacol 29:2639–2643

    Article  PubMed  CAS  Google Scholar 

  10. Abushamaa AM, Sporn TA, Folz RJ (2002) Oxidative stress and inflammation contribute to lung toxicity after a common breast cancer chemotherapy regimen. Am J Physiol Lung Cell Mol Physiol 283:L336–L345

    PubMed  CAS  Google Scholar 

  11. Johnston TP, Montgomery JA (1986) Relationship of structure to anticancer activity and toxicity of the nitrosoureas in animal systems. Cancer Treat Rep 70:13–30

    PubMed  CAS  Google Scholar 

  12. Gibson NW, Hickman JA (1982) The role of isocyanates in the toxicity of antitumour haloalkylnitrosoureas. Biochem Pharmacol 31:2795–2800

    Article  PubMed  CAS  Google Scholar 

  13. Rice KP, Penketh PG, Shyam K, Sartorelli AC (2005) Differential inhibition of cellular glutathione reductase activity by isocyanates generated from the antitumor prodrugs cloretazine and BCNU. Biochem Pharmacol 69:1463–1472

    Article  PubMed  CAS  Google Scholar 

  14. Slatter JG, Davis MR, Han DH, Pearson PG, Baillie TA (1993) Studies on the metabolic fate of caracemide, an experimental antitumor agent, in the rat. Evidence for the release of methyl isocyanate in vivo. Chem Res Toxicol 6:335–340

    Article  PubMed  CAS  Google Scholar 

  15. Kestell P, Gledhill AP, Threadgill MD, Gescher A (1986) S-(N-methylcarbamoyl)-N-acetylcysteine: a urinary metabolite of the hepatotoxic experimental antitumour agent N-methylformamide (NSC 3051) in mouse, rat and man. Biochem Pharmacol 35:2283–2286

    Article  PubMed  CAS  Google Scholar 

  16. Moriarty-Craige SE, Jones DP (2004) Extracellular thiols and thiol/disulfide redox in metabolism. Annu Rev Nutr 24:481–509

    Article  PubMed  CAS  Google Scholar 

  17. Meister A (1994) Glutathione–ascorbic acid antioxidant system in animals. J Biol Chem 269:9397–9400

    PubMed  CAS  Google Scholar 

  18. Smith AC, Boyd MR (1984) Preferential effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on pulmonary glutathione reductase and glutathione/glutathione disulfide ratios: possible implications for lung toxicity. J Pharmacol Exp Ther 229:658–663

    PubMed  CAS  Google Scholar 

  19. Holmgren A, Bjornstedt M (1995) Thioredoxin and thioredoxin reductase. Methods Enzymol 252:199–208

    Article  PubMed  CAS  Google Scholar 

  20. Mustacich D, Powis G (2000) Thioredoxin reductase. Biochem J 346(Pt 1):1–8

    Article  PubMed  CAS  Google Scholar 

  21. Holmgren A, Lu J (2010) Thioredoxin and thioredoxin reductase: current research with special reference to human disease. Biochem Biophys Res Commun 396:120–124

    Article  PubMed  CAS  Google Scholar 

  22. Powis G, Montfort WR (2001) Properties and biological activities of thioredoxins. Annu Rev Pharmacol Toxicol 41:261–295

    Article  PubMed  CAS  Google Scholar 

  23. Arner ES, Holmgren A (2000) Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem 267:6102–6109

    Article  PubMed  CAS  Google Scholar 

  24. Liu Y, Min W (2002) Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activity-independent manner. Circ Res 90:1259–1266

    Article  PubMed  CAS  Google Scholar 

  25. Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, Kawabata M, Miyazono K, Ichijo H (1998) Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J 17:2596–2606

    Article  PubMed  CAS  Google Scholar 

  26. Pennington JD, Jacobs KM, Sun L, Bar-Sela G, Mishra M, Gius D (2007) Thioredoxin and thioredoxin reductase as redox-sensitive molecular targets for cancer therapy. Curr Pharm Des 13:3368–3377

    Article  PubMed  CAS  Google Scholar 

  27. Urig S, Becker K (2006) On the potential of thioredoxin reductase inhibitors for cancer therapy. Semin Cancer Biol 16:452–465

    Article  PubMed  CAS  Google Scholar 

  28. Zeng HH, Wang LH (2010) Targeting thioredoxin reductase: anticancer agents and chemopreventive compounds. Med Chem 6:286–297

    Article  PubMed  CAS  Google Scholar 

  29. Arner ES, Holmgren A (2006) The thioredoxin system in cancer. Semin Cancer Biol 16:420–426

    Article  PubMed  CAS  Google Scholar 

  30. Conrad M (2009) Transgenic mouse models for the vital selenoenzymes cytosolic thioredoxin reductase, mitochondrial thioredoxin reductase and glutathione peroxidase 4. Biochim Biophys Acta 1790:1575–1585

    Article  PubMed  CAS  Google Scholar 

  31. Yoo MH, Xu XM, Carlson BA, Gladyshev VN, Hatfield DL (2006) Thioredoxin reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung carcinoma cells. J Biol Chem 281:13005–13008

    Article  PubMed  CAS  Google Scholar 

  32. Turanov AA, Kehr S, Marino SM, Yoo MH, Carlson BA, Hatfield DL, Gladyshev VN (2010) Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets. Biochem J 430:285–293

    Article  PubMed  CAS  Google Scholar 

  33. Mandal PK, Schneider M, Kolle P, Kuhlencordt P, Forster H, Beck H, Bornkamm GW, Conrad M (2010) Loss of thioredoxin reductase 1 renders tumors highly susceptible to pharmacologic glutathione deprivation. Cancer Res 70:9505–9514

    Article  PubMed  CAS  Google Scholar 

  34. Anestal K, Prast-Nielsen S, Cenas N, Arner ES (2008) Cell death by SecTRAPs: thioredoxin reductase as a prooxidant killer of cells. PLoS ONE 3:1–16

    Article  Google Scholar 

  35. Williams CH, Arscott LD, Muller S, Lennon BW, Ludwig ML, Wang PF, Veine DM, Becker K, Schirmer RH (2000) Thioredoxin reductase two modes of catalysis have evolved. Eur J Biochem 267:6110–6117

    Article  PubMed  CAS  Google Scholar 

  36. Cheng Q, Sandalova T, Lindqvist Y, Arner ES (2009) Crystal structure and catalysis of the selenoprotein thioredoxin reductase 1. J Biol Chem 284:3998–4008

    Article  PubMed  CAS  Google Scholar 

  37. Nordberg J, Zhong L, Holmgren A, Arner ES (1998) Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohalobenzenes by alkylation of both the redox active selenocysteine and its neighboring cysteine residue. J Biol Chem 273:10835–10842

    Article  PubMed  CAS  Google Scholar 

  38. Papp LV, Lu J, Holmgren A, Khanna KK (2007) From selenium to selenoproteins: synthesis, identity, and their role in human health. Antioxid Redox Signal 9:775–806

    Article  PubMed  CAS  Google Scholar 

  39. Witte AB, Anestal K, Jerremalm E, Ehrsson H, Arner ES (2005) Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds. Free Radic Biol Med 39:696–703

    Article  PubMed  CAS  Google Scholar 

  40. Shyam K, Penketh PG, Divo AA, Loomis RH, Patton CL, Sartorelli AC (1990) Synthesis and evaluation of 1,2,2-tris(sulfonyl)hydrazines as antineoplastic and trypanocidal agents. J Med Chem 33:2259–2264

    Article  PubMed  CAS  Google Scholar 

  41. Shyam K, Penketh PG, Loomis RH, Rose WC, Sartorelli AC (1996) Antitumor 2-(aminocarbonyl)-1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-hydrazines. J Med Chem 39:796–801

    Article  PubMed  CAS  Google Scholar 

  42. Kettenbach AN, Gerber SA (2011) Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments. Anal Chem 83:7635–7644

    Article  PubMed  CAS  Google Scholar 

  43. Kettenbach AN, Schweppe DK, Faherty BK, Pechenick D, Pletnev AA, Gerber SA (2011) Quantitative phosphoproteomics identifies substrates and functional modules of aurora and polo-like kinase activities in mitotic cells. Sci Signal 4:rs5

    Article  PubMed  CAS  Google Scholar 

  44. Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database. J Am Soc Mass Spectrom 5:976–989

    Article  CAS  Google Scholar 

  45. Faherty BK, Gerber SA (2010) MacroSEQUEST: efficient candidate-centric searching and high-resolution correlation analysis for large-scale proteomics data sets. Anal Chem 82:6821–6829

    Article  PubMed  CAS  Google Scholar 

  46. Schallreuter KU, Gleason FK, Wood JM (1990) The mechanism of action of the nitrosourea anti-tumor drugs on thioredoxin reductase, glutathione reductase and ribonucleotide reductase. Biochim Biophys Acta 1054:14–20

    Article  PubMed  CAS  Google Scholar 

  47. Seyfried J, Wullner U (2007) Inhibition of thioredoxin reductase induces apoptosis in neuronal cell lines: role of glutathione and the MKK4/JNK pathway. Biochem Biophys Res Commun 359:759–764

    Article  PubMed  CAS  Google Scholar 

  48. Lan L, Zhao F, Wang Y, Zeng H (2007) The mechanism of apoptosis induced by a novel thioredoxin reductase inhibitor in A549 cells: possible involvement of nuclear factor-kappaB-dependent pathway. Eur J Pharmacol 555:83–92

    Article  PubMed  CAS  Google Scholar 

  49. Marzano C, Gandin V, Folda A, Scutari G, Bindoli A, Rigobello MP (2007) Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells. Free Radic Biol Med 42:872–881

    Article  PubMed  CAS  Google Scholar 

  50. Chin L, Andersen JN, Futreal PA (2011) Cancer genomics: from discovery science to personalized medicine. Nat Med 17:297–303

    Article  PubMed  CAS  Google Scholar 

  51. Atkinson JM, Shelat AA, Carcaboso AM, Kranenburg TA, Arnold LA, Boulos N, Wright K, Johnson RA, Poppleton H, Mohankumar KM, Feau C, Phoenix T, Gibson P, Zhu L, Tong Y, Eden C, Ellison DW, Priebe W, Koul D, Yung WK, Gajjar A, Stewart CF, Guy RK, Gilbertson RJ (2011) An integrated in vitro and in vivo high-throughput screen identifies treatment leads for ependymoma. Cancer Cell 20:384–399

    Article  PubMed  CAS  Google Scholar 

  52. Bednar F, Simeone DM (2012) Metformin and cancer stem cells: old drug, new targets. Cancer Prev Res (Phila) 5:351–354

    Article  CAS  Google Scholar 

  53. Arteaga CL, Baselga J (2012) Impact of genomics on personalized cancer medicine. Clin Cancer Res 18:612–618

    Article  PubMed  CAS  Google Scholar 

  54. Lin SH, Kleinberg LR (2008) Carmustine wafers: localized delivery of chemotherapeutic agents in CNS malignancies. Expert Rev Anticancer Ther 8:343–359

    Article  PubMed  CAS  Google Scholar 

  55. Penketh PG, Shyam K, Sartorelli AC (2000) Comparison of DNA lesions produced by tumor-inhibitory 1,2-bis(sulfonyl)hydrazines and chloroethylnitrosoureas. Biochem Pharmacol 59:283–291

    Article  PubMed  CAS  Google Scholar 

  56. Murren J, Modiano M, Kummar S, Clairmont C, Egorin M, Chu E, Sznol M (2005) A phase I and pharmacokinetic study of VNP40101 M, a new alkylating agent, in patients with advanced or metastatic cancer. Invest New Drugs 23:123–135

    Article  PubMed  CAS  Google Scholar 

  57. Raizer J, Rice L, Rademaker A, Chandler J, Levy R, Muro K, Grimm S (2011) A phase I trial of laromustine (VNP40101 M) and temozolomide for patients with malignant gliomas in first relapse or progression. Neuro Oncol 13:60–61

    Google Scholar 

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Acknowledgments

This project was supported by grants from the National Center for Research Resources (5P20RR016463-12 to K.P.R. and P20-RR018787 to S.A.G) and the National Institute of General Medical Sciences (8 P20 GM103423-12 to K.P.R.), of the National Institutes of Health, and from the Colby College Division of Natural Sciences. The authors would also like to acknowledge Alan Sartorelli (Yale University School of Medicine) for furnishing the compounds necessary to begin this project. The authors would like to thank Jeffrey Katz and Nicholas Bizer (Colby College) for providing counsel toward the chemical syntheses.

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Authors and Affiliations

  1. Department of Chemistry, Colby College, 5763 Mayflower Hill Road, Waterville, ME, 04901, USA

    Kevin P. Rice, Edmund J. Klinkerch, Tyler R. Schleicher, Tara J. Kraus & Christopher M. Buros

  2. Department of Genetics and Biochemistry, Geisel School of Medicine at Dartmouth, One Medical Center Drive, Rubin 7th, HB-7937, Lebanon, NH, 03756, USA

    Scott A. Gerber

  3. Department of Molecular and Cell Biology, University of Connecticut, 91 North Eagleville Road, Storrs, CT, 06269, USA

    Tyler R. Schleicher

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  1. Kevin P. Rice
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Correspondence to Kevin P. Rice.

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Rice, K.P., Klinkerch, E.J., Gerber, S.A. et al. Thioredoxin reductase is inhibited by the carbamoylating activity of the anticancer sulfonylhydrazine drug laromustine. Mol Cell Biochem 370, 199–207 (2012). https://doi.org/10.1007/s11010-012-1411-y

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