Skip to main content
SpringerLink
Log in

Targeting ROS overgeneration by N-benzyl-2-nitro-1-imidazole-acetamide as a potential therapeutic reposition approach for cancer therapy

  • PHASE I STUDIES
  • Published:
Investigational New Drugs Aims and scope Submit manuscript

Summary

BackgroundWe investigated the role of reactive oxygen species (ROS) in the anticancer mechanism of N-benzyl-2-nitro-1-imidazole-acetamide (BZN), a drug used in Chagas’ disease treatment. MethodsBALB/c mice, inoculated with Ehrlich ascites carcinoma (EAC), were treated with BZN or BZN + Nacylcysteine (NAC) or NAC for 9 days. Subsequently, the inhibition of tumor growth and angiogenesis as well as animal survival were evaluated. Apoptosis and the cell cycle were evaluated using fluorescence microscopy and flow cytometry, while oxidative stress was evaluated by measuring TBARS content, DNA damage, calcium influx and ROS generation and antioxidant defenses (CAT, SOD, GPx, GST and GR). Immunoblotting was used to evaluate key death and cell cycle proteins. Results BZN treatment inhibited tumor progression (79%), angiogenesis (2.8-fold) and increased animal survival (29%). Moreover, BZN increased ROS levels (42%), calcium influx (55%), TBARS contents (1.9-fold), SOD (4.4-fold), GPx (17.5-fold) and GST (3-fold) activities and GSH depletion (2.5-fold) also caused DNA fragmentation (7.6-fold), increased cleaved PARP and promoted the trapping of cells in the G1 phase, as corroborated by the reduction in cyclin A and increased CDK2 protein levels. In silico DNA and molecular dynamic simulations showed H-bonds and hydrophobic interactions that were confirmed by circular dichroism. Increased apoptosis (232%), induced by treatment with BZN, was demonstrated by apoptotic cell staining and p53 level. Conclusion The current findings indicate that BZN acts as a tumor growth inhibitor and anti-angiogenic agent by ROS overgeneration, which interact with DNA causing damage and triggering apoptosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

Similar content being viewed by others

Abbreviations

CAT:

Catalase

CDK2:

Cyclin-dependentkinase 2

BZN:

Benznidazole (N-benzyl-2-nitro-1- imidazole-acetamide)

GPx:

Glutathione Peroxidase

GROMACS:

Groningen Machine for Chemical Simulations

GSH:

Reduced Glutathione

GST:

Glutathione S-transferase

NAC:

N-Acetyl-L-cysteine

PARP:

Poly (ADP-ribose) polymerase

ROS:

Reactive Oxygen Species

SOD:

Superoxide Dismutase

TBARS:

Thiobarbituric Acid Reactive Substances

References

  1. Pedrosa RC, De Bem AF, Locatelli C, Pedrosa RC, Geremias R, Wilhelm Filho D (2001) Timedependent oxidative stress caused by benzonidazole. Redox Rep 4:265–270

    Article  Google Scholar 

  2. Halliwell B, Gutteridge JMC (2007) Free radicals in biology and medicine. 4thed. Oxford, Clarendon.

  3. Pawlowska N, Gornowicz A, Bielawska A, Surazynski A, Szymanowska A, Czarnomysy R, Bielawski K (2018) The molecular mechanism of anticancer action of novel octahydropyrazino [2,1a:5,4-a´] diisoquinoline derivatives in human gastric cancer cells. Invest. New Drugs 36:970984. https://doi.org/10.1007/s10637-018-0584-y

    Article  CAS  Google Scholar 

  4. Mantindale JL, Holbrook NJ (2002) Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol 192:1–15

    Article  Google Scholar 

  5. Wang J, Yi J (2008) Cancer cell killing via ROS: to increase or decrease, that is the question. Cancer Biol Ther 12:1875–1884. https://doi.org/10.4161/cbt.7.12.70.67

    Article  Google Scholar 

  6. Morton DB (2000) A systematic approach for establishing humane endpoints. ILAR j (2):80–86

  7. Wallace J (2000) Humane Endpoints and Cancer Research. ILAR j (2):87–93

  8. Santra A, Chowdhury A, Ghatak S, Biswas A, Dhali GK (2007) Arsenic induces apoptosis mouse liver is mitochondria dependent and is abrogated by N-acetylcysteine. Toxicol Appl Pharmacol (2):146155. https://doi.org/10.1016/j.taap.2006.12.029

  9. Meier P, Kaplan EL (1958) Nonparametric estimation from incomplete observations. J Am Stat Assoc (282):457–481

  10. Agrawal SS, Saraswatis S, Mathur R, Pandey M (2011) Antitumor properties of boswellic acid against Ehrlich ascites cells bearing mouse. Food Chem Toxicol (9):1924–1934. https://doi.org/10.1016/j.fct.2011.04.007

  11. Mascotti K, McCullough J, Burger SR (2000) HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion (6):693–696

  12. Nunez R (2001) DNA Measurement and Cell Cycle Analysis by Flow Cytometry. Curr Issues Mol Biol 3:67–70

    CAS  PubMed  Google Scholar 

  13. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem (2):351–358

  14. Beutler E, Duron O, Kelly BM (1963) Improved method for the determination of blood glutathione. J Lab Clin Med 6:882–888

    Google Scholar 

  15. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126

    Article  CAS  Google Scholar 

  16. Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175

    CAS  PubMed  Google Scholar 

  17. Flohé L, Gunzler WA (1984) Assays of glutathione peroxidase. Methods Enzymol 105:114–121

    Article  Google Scholar 

  18. Habig WH, Pabst MJ, Jakoby WB (1976) Glutathione S-transferases, The first enzymatic step in mercapturic acid formation. J Biol Chem 22:7130–7139

    Google Scholar 

  19. Calberg I, Mannervik B (1985) Glutathione reductase from rat liver. Methods Enzymol 113:484490

    Google Scholar 

  20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 1:265–275

    Google Scholar 

  21. Basu A, Ghosh P, Bhattacharjee A, Patra AR, Bhattacharya S (2015) Prevention of myelosuppression and genotoxicity induced by cisplatin in murine bone marrow cells: effect of an organovanadium compound vanadium (III)-Lcysteine. Mutagenesis (4):509–517. https://doi.org/10.1093/mutage/gev011

  22. Batra S, Jogren CS (1983) Effect of estrogen treatment on calcium uptake by the rat uterine smooth muscle. Life Sci (4):315–319

  23. Frederico MJS, Castro AJG, Mascarello A, Mendes CP, Kappel VD, Stumpf TR, Leal PC, Nunes RJ, Yunes RA, Silva FRMB (2012) Acylhydrazones contribute to serum glucose homeostasis through dual physiological targets. Curr Top Med Chem 19:2049–2058

    Article  Google Scholar 

  24. Singh NP, McCoy MT, Tice RR, Schneider EL (1998) A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1:184–191

    Google Scholar 

  25. Laemmli UK (1970) Cleavage of structural protein during assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  26. Li JH, Wang JT, Hu P, Zhang LY, Chen ZN, Mao ZW, Ji LN (2008) Synthesis, structure and nuclease activity of copper complexes of bisubstituted 2,2′-bipyridine ligands bearing ammonium groups. Polyhedron 27:1898–1904. https://doi.org/10.1016/j.poly.2008.02.032

    Article  CAS  Google Scholar 

  27. Bortolotto T, Silva-Caldeira PP, Pich CT, Pereira-Maia EC, Terenzi H (2016) Tun able DNA cleavage activity promoted by copper (II) ternary complexes with N-donor heterocyclic ligands. Chem Common (44):7130–7133. https://doi.org/10.1039/C6CC03142K

  28. O’Boyle N, Banck M, James C, Morley C, Vandermeersch T, Hutchison G (2011) Open Babel: An open chemical toolbox. J. Cheminf:30–33. https://doi.org/10.1186/1758-2946-3-33

  29. Wang J, Wang W, Kollman PA, Case DA (2006) Automatic atom type and bond type perception in molecular mechanical calculations. J Mol Graph Model (2):247–260. https://doi.org/10.1016/j.jmgm.2005.12.005

  30. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem (13):1605–1612. https://doi.org/10.1002/jcc.20084

  31. McElfresh GW, Deligkaris C (2018) A vibrational entropy term for DNA docking with autodock. Comput BiolChem 74:286–293. https://doi.org/10.1016/j,compbiolchem.2018.03.027

    Article  CAS  Google Scholar 

  32. Forli S, Huey R, Pique ME, Sanner M, Goodsell DS, Olson AJ (2016) Computational proteinligand docking and virtual drug screening with the AutoDock suite. Nat Protoc 5:905–919. https://doi.org/10.1038/improt.2016.051

    Article  Google Scholar 

  33. Trott O, Olson AJ (2010) Auto Dock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J Comput Chem 2:455–461. https://doi.org/10.1002/jcc.21334

    Article  CAS  Google Scholar 

  34. Schrödinger LLC (2015) The PyMOL Molecular Graphics System, Version 1.8, Schrödinger, L.C.C., 2015

  35. Laskowski RA, Swindells MB (2011) LigPlot+: Multiple LigandProtein Interaction Diagrams for Drug Discovery. J Chem Inf Model 51:2778–2786. https://doi.org/10.1021/cj200227u

    Article  CAS  Google Scholar 

  36. Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR (2012) Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminformatics 4:117. https://doi.org/10.1186/1758-2946-4-17

    Article  CAS  Google Scholar 

  37. Van der SpoelD, Lindahl E, Hess B, Kutzner C, Van Buuren AR, Apol E, Meulenhoff PJ, Tieleman DP, Sijbers ALTM, Feenstra KA, Van Drunen R, Berendsen HJC (2009) GROMACS User Manual version 4.0.[S.l.], 1–326

  38. Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C (2006) Comparison of multiple AMBER force fields and development of improved protein backbone parameters. Proteins 15:712–725. https://doi.org/10.1002/prot.21123

    Article  CAS  Google Scholar 

  39. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys (2):926–935

  40. Kumari R, Kumar R, Open Source Drug Discovery Consortium, Lynn A (2014) g_MMPBSA--a GROMACS tool for high-throughput MM-PBSA calculations. J ChemI nf Model 54:1951–1962. https://doi.org/10.1021/ci500020m

    Article  CAS  Google Scholar 

  41. Hsin J, Arkhipov A, Yin Y, Stone JE, Schulten K (2008) Using VMD - An Introductory Tutorial. Curr Protoc Bioinformatics. December; Chapter:Unit–5.7. https://doi.org/10.1002/0471250953.bi0507s24

  42. Verlet L (1967) Computer "Experiments" on Classical Fluids. I. Thermodynamical Properties of Lennard-Jones Molecules. Phys Rev 159:98–103

    Article  CAS  Google Scholar 

  43. Lemkul JA, Allen WJ, Bevan DR (2010) Practical Considerations for Building GROMOSCompatible Small-Molecule Topologies. J Chem Inf Model 50:2221–2235. https://doi.org/10.1021/ci100335w

    Article  CAS  PubMed  Google Scholar 

  44. Ganeshpandian M, Loganathan R, Ramakrishnan S, Riyasdeen A, Akbarsha MA, Palaniandavar M (2013) Interaction of mixed ligand copper (II) complexes with CT DNA and BSA: Effect of primary ligand hydrophobicity on DNA and protein binding cleavage and anticancer activities. Polyhedron 52:924–938. https://doi.org/10.1016/j.poly.2012.07.021

    Article  CAS  Google Scholar 

  45. Galadari S, Rahman A, Pallichankandy S, Thayyullathil F (2017) Reactive oxygen species and cancer paradox: to promote or to suppress? Free Radic Biol Med 104:144–164. https://doi.org/10.1016/j.freeradbiomed.2017.01.004

    Article  CAS  PubMed  Google Scholar 

  46. Adams JM, Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281:1322–1326

    Article  CAS  Google Scholar 

  47. Calvo KL, Ronco MT, Noguera NI, Garcia F (2013) Benznidazole modulates cell proliferation in acute leukemia cells. Immunopharmacol. Immunotoxicol (4):478–486. https://doi.org/10.3109/08923973.2013.811597

  48. Díaz de Toranzo EG, Castro JA, Franke de Cazzulo BM, Cazzulo JJ (1988) Interaction of benznidazole reactive metabolites with nuclear and kinetoplastic DNA, proteins and lipids from Trypanosoma cruzi. Experientia 44:880–881

    Article  Google Scholar 

  49. Hempel N, Trebak M (2017) Crosstalk between calcium and reactive oxygen species signaling in cancer. Cell. Calcium 63:70–98. https://doi.org/10.1016/j.ceca.2017.01.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Rao VR, Perez-Nuut M, Kaja S, Genyile S (2015)Voltage-gated ion channels in cancer cell proliferation. Cancers (2):849–875. https://doi.org/10.3390/cancers7020813

  51. Gogvadze V, Robertson JD, Zhivotovsky B, Orrenius S (2001) Cytochrome c release occurs via Ca2+-dependent and Ca2+-independent mechanisms that are regulated by Bax. J Biol Chem (22):19066–19071. https://doi.org/10.1074/jbc.M100614200

  52. Dubrez L, Coll JL, Hurbin A, Solary E, Favrot MC (2001) Caffeine sensitizes human H358 cell line to p53-mediated apoptosis by inducing mitochondrial translocation and conformational change of BAX protein. J Biol Chem 42:38980–38987. https://doi.org/10.1074/jbc.M100614200

    Article  Google Scholar 

  53. El Hassan M, Calladine CR (1996)Propeller-Twisting of Base-pairs and the Conformational Mobility of Dinucleotide Steps in DNA. J Mol Biol 259:95–103

    Article  Google Scholar 

  54. Mann J, Baron A, Yaw OB, Eric J, Gary P, Lloyd RK, Stephen N (2001) A new class of symmetric bisbenzimidazole-based DNA minor groove-binding agents showing antitumor activity. J Med Chen 44:138–144

    Article  CAS  Google Scholar 

  55. Wilson WD, Tanious FA, Mathis A, Tevis D, Hall JE, Boykin DW (2008) Antiparasitic compounds that target DNA. Biochimie 90:999–1014. https://doi.org/10.1016/j.biochi.2008.02.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Qi R, Wang Q, Ren P (2016) General van der Waals potential for common organic molecules. Bioorg Med Chem 24:4911–4919. https://doi.org/10.1016/j.bmc.2016.07.062

    Article  PubMed  PubMed Central  Google Scholar 

  57. Ussery DW (2002) DNA structure A-, B- and Z-DNA helix families. Encyclopedia of life sciences. John Wiley & Sons, Ltd. Chichester, U.K.,2002.

  58. Morriss-Andrews A, Rottler J, Plotkin SS (2010) A systematically coarse-grained model for DNA and its predictions for persistence length, stacking, twist, and chirality. J Chem Phys 132:1–19. https://doi.org/10.1063/1.3269994

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors are grateful to CEBIME-UFSC and for the financial support provided by the Brazilian governamental agencies Conselho Nacional de Pesquisa (CNPq) and from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brazil. RCP (Proc. 302404/2017-2) and DWF (Proc. 303234/2015-6) are recipients of research grants from CNPq, Brazil. RCZ, NSRSM and VMASG are fellows of CAPES/CNPq, Brazil.

Funding

The work was supported by the Brazilian governamental agencies to research Conselho Nacional de Pesquisa (CNPq) and from Coordenação de Aperfeiçoamento de Pessoal de.

Nível Superior (CAPES).

Author information

Authors and Affiliations

  1. Biochemistry Department, Federal University of Santa Catarina (UFSC), Florianópolis, SC, Brazil

    Rodrigo C. Zeferino, Nádia S. R. S. Mota, Valdelúcia M. A. S. Grinevicius, Paola M. Sulis, Fátima R. M. B. Silva & Rozangela C. Pedrosa

  2. Department of Clinical Analysis, Federal University of Paraná (UFPR), Curitiba, PR, Brazil

    Karina B. Filipe

  3. Ecology and Zoology Department, Federal University of Santa Catarina (UFSC), Florianópolis, SC, Brazil

    Danilo W. Filho

  4. Center for Sciences, Technologies and Health, Federal University of Santa Catarina (UFSC), Araranguá, SC, Brazil

    Claus T. Pich

Authors
  1. Rodrigo C. Zeferino
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nádia S. R. S. Mota
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Valdelúcia M. A. S. Grinevicius
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karina B. Filipe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paola M. Sulis
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fátima R. M. B. Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Danilo W. Filho
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Claus T. Pich
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Rozangela C. Pedrosa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rozangela C. Pedrosa.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest. Author Rodrigo C. Zeferino declares that he has no conflict of interest. Author Nádia S.R.S Mota declares that she has no conflict of interest. Author Valdelúcia M.A.S. Grinevicius declares that she has no conflict of interest. Author Karina B. Filipe declares that she has no conflict of interest. Author Paola M. Sulis declares that she has no conflict of interest. Author Fátima R.M.B Silva declares that she has no conflict of interest. Author Danilo W. Filho declares that he has no conflict of interest. Author Claus T. Pich declares that he has no conflict of interest. Author Rozangela C. Pedrosa declares that she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The animal study followed the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised in 1978) and the experimental protocol was approved by the local Ethics Committee on Animal Use (CEUA; UFSC PP00784).

Informed consent

Informed consent was obtained from all individual participants included in the study.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Fig. 1

N-benzyl-2-nitro-1-imidazole-acetamide (benznidazole, BZN) chemical structure and metabolization route (PNG 39 kb)

High Resolution (TIF 336 kb)

Supplementary Fig. 2

– BZN binding to DNA: (a) Number of H-bonds formed throughout the MD simulation between BZN and da17 nucleotide residue. (b) BZN and da17 dihedral angles distribution (average angle 146.251o). (c) Binding energy (ΔE) of complex calculated with MM-PBSA. (d) Molecular Mechanics Energy calculated in vacuum with major contribution of van der Waals Energy (blue line) for Total Energy (red line). (e) Contributions to Total Energy related to each residue of nucleotide of DNA (1–24) and ligand (BZN) (25); insert shows color score for contribution energies of da17 (light blue) and BZN (red). (f) Complex Solvation Free Energy (ΔG solv) related to SASA calculated with MM-PBSA (PNG 192 kb)

High Resolution (TIF 357 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zeferino, R.C., Mota, N.S.R.S., Grinevicius, V.M.A.S. et al. Targeting ROS overgeneration by N-benzyl-2-nitro-1-imidazole-acetamide as a potential therapeutic reposition approach for cancer therapy. Invest New Drugs 38, 785–799 (2020). https://doi.org/10.1007/s10637-019-00820-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10637-019-00820-5

Keywords

Search

Navigation