Targeting tumor hypoxia with the epigenetic anticancer agent, RRx-001: a superagonist of nitric oxide generation

Abstract

This study reveals a novel interaction between deoxyhemoglobin, nitrite and the non-toxic compound, RRx-001, to generate supraphysiologic levels of nitric oxide (NO) in blood. We characterize the nitrite reductase activity of deoxyhemoglobin, which in the presence of bound RRx-001 reduces nitrite at a much faster rate, leading to markedly increased NO generation. These data expand on the paradigm that hemoglobin generates NO via nitrite reduction during hypoxia and ischemia when nitric oxide synthase (NOS) function is limited. Here, we demonstrate that RRx-001 greatly enhances NO generation from nitrite reduction. RRx-001 is thus the first example of a functional superagonist for nitrite reductase. We hypothesize that physiologically this reaction releases the potentially cytotoxic effector NO selectively in hypoxic tumor regions. It may be that a binary NO–H2O2 trigger is indirectly responsible for the observed tumoricidal activity of RRx-001 since NO is known to inhibit mitochondrial respiration.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. 1.

    Moroz LL, Kohn AB. Parallel evolution of nitric oxide signaling: diversity of synthesis and memory pathways. Front Biosci. 2011;16:2008–51.

    CAS  Article  Google Scholar 

  2. 2.

    Crawford JH, Isbell TS, Huang Z, Shiva S, Chacko BK, Schechter AN, Darley-Usmar VM, Kerby JD, Lang JD Jr, Kraus D, Ho C, Gladwin MT, Patel RP. Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation. Blood. 2006;107:566–74. doi:10.1182/blood-2005-07-2668.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Huang Z, Shiva S, Kim-Shapiro DB, Patel RP, Ringwood LA, Irby CE, Huang KT, Ho C, Hogg N, Schechter AN, Gladwin MT. Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control. J Clin Invest. 2005;115:2099–107. doi:10.1172/JCI24650.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Oronsky BT, Knox SJ, Scicinski JJ. Is Nitric Oxide (NO) the Last Word in Radiosensitization? A review. Transl Oncol. 2012;5:66–71.

    Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Mocellin S, Bronte V, Nitti D. Nitric oxide, a double edged sword in cancer biology: searching for therapeutic opportunities. Med Res Rev. 2007;27:317–52. doi:10.1002/med.20092.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Oronsky B, Fanger GR, Oronsky N, Knox S, Scicinski J. The implications of hyponitroxia in cancer. Transl Oncol. 2014;7:167–73. doi:10.1016/j.tranon.2014.02.001.

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Burke AJ, Sullivan FJ, Giles FJ, Glynn SA. The yin and yang of nitric oxide in cancer progression. Carcinogenesis. 2013;34:503–12. doi:10.1093/carcin/bgt034.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Scicinski J, Oronsky B, Ning S, Knox S, Peehl D, Kim MM, Langecker P, Fanger G. NO to cancer: the complex and multifaceted role of nitric oxide and the epigenetic nitric oxide donor, RRx-001. Redox Biol. 2015;6:1–8. doi:10.1016/j.redox.2015.07.002.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol. 1996;271:C1424–37.

    CAS  PubMed  Google Scholar 

  10. 10.

    Ning S, Bednarski M, Oronsky B, Scicinski J, Saul G, Knox SJ. Dinitroazetidines are a novel class of anticancer agents and hypoxia-activated radiation sensitizers developed from highly energetic materials. Cancer Res. 2012;72:2600–8. doi:10.1158/0008-5472.CAN-11-2303.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Zhao H, Ning S, Scicinski J, Oronsky B, Knox S, Peehl DM. Abstract 3515: RRx-001: a double action systemically non-toxic epigenetic agent for cancer therapy. Cancer Res. 2015;75:3515. doi:10.1158/1538-7445.am2015-3515.

    Article  Google Scholar 

  12. 12.

    Zhao H, Ning S, Scicinski J, Oronsky B, Knox SJ, Peehl DM. Epigenetic effects of RRx-001: a possible unifying mechanism of anticancer activity. Oncotarget. 2015. doi:10.18632/oncotarget.16526.

    Google Scholar 

  13. 13.

    Reid T, Dad S, Korn R, Oronsky B, Knox S, Scicinski J. two case reports of resensitization to previous chemotherapy with the novel hypoxia-activated hypomethylating anticancer agent RRx-001 in metastatic colorectal cancer patients. Case Rep Oncol. 2014;7:79–85. doi:10.1159/000358382.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Reid T, Oronsky B, Scicinski J, Scribner CL, Knox SJ, Ning S, Peehl DM, Korn R, Stirn M, Carter CA, Oronsky A, Taylor MJ, Fitch WL, Cabrales P, Kim MM, Burris HA 3rd, Lao CD, Abrouk NE, Fanger GR, Infante JR. Safety and activity of RRx-001 in patients with advanced cancer: a first-in-human, open-label, dose-escalation phase 1 study. Lancet Oncol. 2015;16:1133–42. doi:10.1016/S1470-2045(15)00089-3.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Scicinski J, Oronsky B, Fitch W, Taylor M, Luo G, Musick T, Marini J, Adams C, Schicker M, Gohdes M, McKenzie D, Ridgewell R. Disposition of 14C-RRx-001 in rats after a single intravenous administration and in blood from rats, dogs, monkeys, and humans. In: ISSX Annual meeting 2011, p. Abstract P81; 2011.

  16. 16.

    Scicinski J, Oronsky B, Taylor M, Luo G, Musick T, Marini J, Adams CM, Fitch WL. Preclinical evaluation of the metabolism and disposition of RRx-001, a novel investigative anticancer agent. Drug Metab Dispos. 2012;40:1810–6. doi:10.1124/dmd.112.046755.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Scicinski J, Oronsky B, Cooper V, Taylor M, Alexander M, Hadar R, Cosford R, Fleischmann T, Fitch WL. Development of methods for the bioanalysis of RRx-001 and metabolites. Bioanalysis. 2014;6:947–56. doi:10.4155/bio.13.331.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Allen BW, Piantadosi CA. How do red blood cells cause hypoxic vasodilation? The SNO-hemoglobin paradigm. Am J Physiol Heart Circ Physiol. 2006;291:H1507–12. doi:10.1152/ajpheart.00310.2006.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Gladwin MT, Ognibene FP, Pannell LK, Nichols JS, Pease-Fye ME, Shelhamer JH, Schechter AN. Relative role of heme nitrosylation and beta-cysteine 93 nitrosation in the transport and metabolism of nitric oxide by hemoglobin in the human circulation. Proc Natl Acad Sci USA. 2000;97:9943–8. doi:10.1073/pnas.180155397.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Lundberg JO, Gladwin MT, Ahluwalia A, Benjamin N, Bryan NS, Butler A, Cabrales P, Fago A, Feelisch M, Ford PC, Freeman BA, Frenneaux M, Friedman J, Kelm M, Kevil CG, Kim-Shapiro DB, Kozlov AV, Lancaster JR Jr, Lefer DJ, McColl K, McCurry K, Patel RP, Petersson J, Rassaf T, Reutov VP, Richter-Addo GB, Schechter A, Shiva S, Tsuchiya K, van Faassen EE, Webb AJ, Zuckerbraun BS, Zweier JL, Weitzberg E. Nitrate and nitrite in biology, nutrition and therapeutics. Nat Chem Biol. 2009;5:865–9. doi:10.1038/nchembio.260.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Wardman P, Rothkamm K, Folkes LK, Woodcock M, Johnston PJ. Radiosensitization by nitric oxide at low radiation doses. Radiat Res. 2007;167:475–84. doi:10.1667/RR0827.1.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Straessler NA, Lesley MW, Cannizzo LF. development of a safe and efficient two-step synthesis for preparing 1-bromoacetyl-3,3-dinitroazetidine: a novel clinical anticancer candidate. Org Process Res Dev. 2012;16:512–7. doi:10.1021/op2003216.

    CAS  Article  Google Scholar 

  23. 23.

    Asakura T. Automated method for determination of oxygen equilibrium curves of red cell suspensions under controlled buffer conditions and its clinical applications. Crit Care Med. 1979;7:391–5.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Kuypers FA, Scott MD, Schott MA, Lubin B, Chiu DT. Use of ektacytometry to determine red cell susceptibility to oxidative stress. J Lab Clin Med. 1990;116:535–45.

    CAS  PubMed  Google Scholar 

  25. 25.

    Morris CR, Suh JH, Hagar W, Larkin S, Bland DA, Steinberg MH, Vichinsky EP, Shigenaga M, Ames B, Kuypers FA, Klings ES. Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease. Blood. 2008;111:402–10. doi:10.1182/blood-2007-04-081703.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Fens MH, Larkin SK, Oronsky B, Scicinski J, Morris CR, Kuypers FA. The capacity of red blood cells to reduce nitrite determines nitric oxide generation under hypoxic conditions. PLoS ONE. 2014;9:e101626. doi:10.1371/journal.pone.0101626.

    Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982;126:131–8.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Lang F, Lang KS, Lang PA, Huber SM, Wieder T. Mechanisms and significance of eryptosis. Antioxid Redox Signal. 2006;8:1183–92. doi:10.1089/ars.2006.8.1183.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Comfurius P, Senden JM, Tilly RH, Schroit AJ, Bevers EM, Zwaal RF. Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase. Biochim Biophys Acta. 1990;1026:153–60.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Rand ML, Wang H, Pluthero FG, Stafford AR, Ni R, Vaezzadeh N, Allison AC, Kahr WH, Weitz JI, Gross PL. Diannexin, an annexin A5 homodimer, binds phosphatidylserine with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation. J Thromb Haemost. 2012;10:1109–19. doi:10.1111/j.1538-7836.2012.04716.x.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Singel DJ, Stamler JS. Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitrosohemoglobin. Annu Rev Physiol. 2005;67:99–145. doi:10.1146/annurev.physiol.67.060603.090918.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Bunn HF, Forget B. Hemoglobin: molecular, genetic and clinical aspects. Philadelphia: W.B. Saunders Company; 1986.

    Google Scholar 

  33. 33.

    Thomas JA, Mallis RJ, Sies H. Protein S-thiolation, S-nitrosylation, and irreversible sulfhydryl oxidation: roles in redox regulation. In: Gitler C, Danon A, editors. Cellular implications of redox signaling. London: Imperial College Press; 2003. p. 141–74.

    Google Scholar 

  34. 34.

    Park S, Hayes BL, Marankan F, Mulhearn DC, Wanna L, Mesecar AD, Santarsiero BD, Johnson ME, Venton DL. Regioselective covalent modification of hemoglobin in search of antisickling agents. J Med Chem. 2003;46:936–53. doi:10.1021/jm020361k.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Gladwin MT, Kim-Shapiro DB. The functional nitrite reductase activity of the heme-globins. Blood. 2008;112:2636–47. doi:10.1182/blood-2008-01-115261.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Schulze A, Harris AL. How cancer metabolism is tuned for proliferation and vulnerable to disruption. Nature. 2012;491:364–73. doi:10.1038/nature11706.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Zhang R, Kang KA, Kim KC, Na SY, Chang WY, Kim GY, Kim HS, Hyun JW. Oxidative stress causes epigenetic alteration of CDX1 expression in colorectal cancer cells. Gene. 2013;524:214–9. doi:10.1016/j.gene.2013.04.024.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Acharya A, Das I, Chandhok D, Saha T. Redox regulation in cancer: a double-edged sword with therapeutic potential. Oxid Med Cell Longev. 2010;3:23–34. doi:10.4161/oxim.3.1.10095.

    Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Oronsky BT, Reid T, Knox SJ, Scicinski JJ. The scarlet letter of alkylation: a mini review of selective alkylating agents. Transl Oncol. 2012;5:226–9.

    Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Hirst DG, Robson T. Nitrosative stress in cancer therapy. Front Biosci. 2007;12:3406–18.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Hirst DG, Robson T. Nitrosative stress as a mediator of apoptosis: implications for cancer therapy. Curr Pharm Des. 2010;16:45–55.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007;87:315–424. doi:10.1152/physrev.00029.2006.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Oronsky BT, Knox SJ, Scicinski J. Six degrees of separation: the oxygen effect in the development of radiosensitizers. Transl Oncol. 2011;4:189–98.

    Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Oronsky B, Oronsky N, Lybeck M, Fanger F, Scicinski J. Targeting hyponitroxia in cancer therapy. In: Bonavida B, editor. Nitric oxide and cancer: pathogenesis and therapy. New York: Springer International Publishing; 2015. p. 39–48.

    Google Scholar 

  45. 45.

    Ettwig KF, Butler MK, Le Paslier D, Pelletier E, Mangenot S, Kuypers MM, Schreiber F, Dutilh BE, Zedelius J, de Beer D, Gloerich J, Wessels HJ, van Alen T, Luesken F, Wu ML, van de Pas-Schoonen KT, Op den Camp HJ, Janssen-Megens EM, Francoijs KJ, Stunnenberg H, Weissenbach J, Jetten MS, Strous M. Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature. 2010;464:543–8. doi:10.1038/nature08883.

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Richardson DJ. Bacterial respiration: a flexible process for a changing environment. Microbiology. 2000;146(Pt 3):551–71. doi:10.1099/00221287-146-3-551.

    CAS  Article  PubMed  Google Scholar 

Download references

Author’s contributions

The authors state that all authors (MH, PC, JS, SK, JS, FK, NO, ML, AO and BO) contributed to all stages of work behind this manuscript: idea conception, design and execution of the studies, analysis, drafting, writing and editing of the manuscript.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Bryan Oronsky.

Ethics declarations

Disclosure

The authors disclose that EpicentRx Inc. has funded parts of this research.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fens, M.H., Cabrales, P., Scicinski, J. et al. Targeting tumor hypoxia with the epigenetic anticancer agent, RRx-001: a superagonist of nitric oxide generation. Med Oncol 33, 85 (2016). https://doi.org/10.1007/s12032-016-0798-9

Download citation

Keywords

  • Oncology
  • RRx-001
  • Nitric oxide
  • Deoxyhemoglobin
  • Nitric oxide synthase