Expression of Enterocin-P in HEK Platform: Evaluation of Its Cytotoxic Effects on Cancer Cell Lines and Its Potency to Interact with Cell-Surface Glycosaminoglycan by Molecular Modeling

Abstract

Cancer remains one of the leading causes of death worldwide. Introduction of natural compounds with anticancer properties can be an effective step in the prevention and treatment of cancer. Antimicrobial peptides (AMPs) are short peptides whose anticancer activities have been proved previously. In the present study, Enterocin-P (EntP) as a bacteriocin of E. faecium was expressed in HEK expression system by pcDNA3.1 + vector. The recombinant peptide was purified from culture medium using Ni2+ affinity chromatography with an average yield of 0.6 mg/ml. The cytotoxic activity of the recombinant peptide was determined toward some cancer cell lines including: SW1353, HUH7, Huh-7.5, C26, B16F0 and NIH3T3 as a normal cell line. Our results showed that EntP peptide had selective cytotoxicity activity only against C26 (IC50: 0.32 mg/ml) and SW1353 (IC50: 1.36 mg/ml) as cancer cell and did not show cytotoxic properties against normal cell. In the second phase of our study, to better understand the mechanisms of EntP peptide, we have tried to predict the possible interaction of this peptide to predominantly negative charge molecules in the cell membrane of cancer cells. Our in-silico analysis revealed that EntP peptide has strong tendency to chondroitin sulfate (− 189 ± 6.24 kJ/mol) and heparan sulfate (− 115 ± 5.12 kJ/mol) as two well-known anionic molecules on surface cancer cells.

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References

  1. Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E (2015) GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1:19–25

    Article  Google Scholar 

  2. Ankaiah D, Esakkiraj P, Perumal V, Ayyanna R, Venkatesan A (2017) Probiotic characterization of Enterococcus faecium por1: cloning, over expression of enterocin-A and evaluation of antibacterial, anti-cancer properties. J Funct Foods 38:280–292

    CAS  Article  Google Scholar 

  3. Araña MJ, Vallespi MG, Chinea G, Vallespi GV, Rodriguez-Alonso I, Garay HE, Buurman WA, Reyes O (2003) Inhibition of LPS-responses by synthetic peptides derived from LBP associates with the ability of the peptides to block LBP–LPS interaction. J Endotoxin Res 9:281–291

    Google Scholar 

  4. Berendsen HJ, Postma JP, van Gunsteren WF, Hermans J (1981) Interaction models for water in relation to protein hydration. In: Pullman B (ed) Intermolecular forces. Springer, Berlin, pp 331–342

    Google Scholar 

  5. Berendsen HJ, van der Spoel D, van Drunen R (1995) GROMACS: a message-passing parallel molecular dynamics implementation. Comput Phys Commun 91:43–56

    CAS  Article  Google Scholar 

  6. Bhattacharjya S (2016) NMR structures and interactions of antimicrobial peptides with lipopolysaccharide: connecting structures to functions. Curr Top Med Chem 16:4–15

    CAS  PubMed  Article  Google Scholar 

  7. Borrelli A, Tornesello A, Tornesello M, Buonaguro F (2018) Cell penetrating peptides as molecular carriers for anti-cancer agents. Molecules 23:295

    PubMed Central  Article  Google Scholar 

  8. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    CAS  PubMed  Article  Google Scholar 

  9. Cintas LM, Casaus P, Håvarstein LS, Hernandez PE, Nes IF (1997) Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl Environ Microbiol 63:4321–4330

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Cleveland J, Montville TJ, Nes IF, Chikindas ML (2001) Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol 71:1–20

    CAS  PubMed  Article  Google Scholar 

  11. Dobrzyńska I, Szachowicz-Petelska B, Sulkowski S, Figaszewski Z (2005) Changes in electric charge and phospholipids composition in human colorectal cancer cells. Mol Cell Biochem 276:113–119

    PubMed  Article  Google Scholar 

  12. Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103:8577–8593

    CAS  Article  Google Scholar 

  13. Eswar et al., 2006 Eswar N, Webb B, Marti-Renom MA, Madhusudhan M, Eramian D, Shen MY, Pieper U, Sali A (2006) Comparative protein structure modeling using Modeller. Curr Protoc Bioinformatics 15:5.6.1–5.6.30.

  14. Fadnes B, Rekdal Ø, Uhlin-Hansen L (2009) The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells. BMC Cancer 9:183

    PubMed  PubMed Central  Article  Google Scholar 

  15. Gaspar D, Veiga AS, Castanho MA (2013) From antimicrobial to anticancer peptides: a review. Front Microbiol 4:294

    PubMed  PubMed Central  Article  Google Scholar 

  16. Gratia A (1925) Sur un remarquable exemple d'antagonisme entre deux souches de coilbacille. C R Seances Soc Biol Fil 93:1040–1041

    Google Scholar 

  17. Gutiérrez J, Criado R, Citti R, Martín M, Herranz C, Nes I, Cintas L, Hernández P (2005a) Cloning, production and functional expression of enterocin P, a sec-dependent bacteriocin produced by Enterococcus faecium P13, in Escherichia coli. Int J Food Microbiol 103:239–250

    PubMed  Article  Google Scholar 

  18. Gutiérrez J, Criado R, Martín M, Herranz C, Cintas LM, Hernández PE (2005b) Production of enterocin P, an antilisterial pediocin-like bacteriocin from Enterococcus faecium P13, in Pichia pastoris. Antimicrob Agents Chemother 49:3004–3008

    PubMed  PubMed Central  Article  Google Scholar 

  19. Hess B (2008) P-LINCS: A parallel linear constraint solver for molecular simulation. J Chem Theory Comput 4:116–122

    CAS  PubMed  Article  Google Scholar 

  20. Hoskin DW, Ramamoorthy A (2008) Studies on anticancer activities of antimicrobial peptides. Biochim Biophys Acta (BBA) 1778:357–375

    CAS  Article  Google Scholar 

  21. Jordan M, Köhne C, Wurm FM (1998) Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Transfection and protein expression in mammalian cells. Cytotechnology 26:39–47

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Kaur S, Kaur S (2015) Bacteriocins as potential anticancer agents. Front Pharmacol 6:272

    PubMed  PubMed Central  Article  Google Scholar 

  23. Kumari R, Kumar R, Open Source Drug Discovery Consortium, Lynn A (2014) g_mmpbsa—a GROMACS tool for high-throughput MM-PBSA calculations. J Chem Inf Model 54:1951–1962

    Article  Google Scholar 

  24. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291

    CAS  Article  Google Scholar 

  25. Maisnier-Patin S, Forni E, Richard J (1996) Purification, partial characterisation and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese. Int J Food Microbiol 30:255–270

    CAS  PubMed  Article  Google Scholar 

  26. Nes IF, Holo H (2000) Class II antimicrobial peptides from lactic acid bacteria. Pept Sci 55:50–61

    CAS  Article  Google Scholar 

  27. Nosé S, Klein M (1983) Constant pressure molecular dynamics for molecular systems. Mol Phys 50:1055–1076

    Article  Google Scholar 

  28. O’Sullivan L, Ross R, Hill C (2002) Potential of bacteriocin-producing lactic acid bacteria for improvements in food safety and quality. Biochimie 84:593–604

    PubMed  Article  Google Scholar 

  29. Oostenbrink C, Villa A, Mark AE, Van Gunsteren WF (2004) A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6. J Comput Chem 25:1656–1676

    CAS  PubMed  Article  Google Scholar 

  30. Perumal V, Venkatesan A (2017) Antimicrobial, cytotoxic effect and purification of bacteriocin from vancomycin susceptible Enterococcus faecalis and its safety evaluation for probiotization. LWT Food Sci Technol 78:303–310

    CAS  Article  Google Scholar 

  31. Poeta P, Costa D, Rojo-Bezares B, Zarazaga M, Klibi N, Rodrigues J, Torres C (2007) Detection of antimicrobial activities and bacteriocin structural genes in faecal enterococci of wild animals. Microbiol Res 162:257–263

    CAS  PubMed  Article  Google Scholar 

  32. Riedl S, Rinner B, Asslaber M, Schaider H, Walzer S, Novak A, Lohner K, Zweytick D (2011) In search of a novel target—phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy. Biochim Biophys Acta (BBA) 1808:2638–2645

    CAS  Article  Google Scholar 

  33. Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn, vol 3. Cold Springs Harbor Laboratory Press, Cold Springs Harbor.

  34. Schweizer F (2009) Cationic amphiphilic peptides with cancer-selective toxicity. Eur J Pharmacol 625:190–194

    CAS  PubMed  Article  Google Scholar 

  35. Tanhaeian A, Jaafari MR, Ahmadi FS, Vakili-Ghartavol R, Sekhavati MH (2018) Secretory expression of a chimeric peptide in Lactococcus lactis: assessment of its cytotoxic activity and a deep view on its interaction with cell-surface glycosaminoglycans by molecular modeling. Probiotics Antimicrob Proteins 11(3):1034–1041

    Article  Google Scholar 

  36. Tanhaeian A, Damavandi MS, Mansury D, Ghaznini K (2019) Expression in eukaryotic cells and purification of synthetic gene encoding enterocin P: a bacteriocin with broad antimicrobial spectrum. AMB Express 9:6

    PubMed  PubMed Central  Article  Google Scholar 

  37. Utsugi T, Schroit AJ, Connor J, Bucana CD, Fidler IJ (1991) Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res 51:3062–3066

    CAS  PubMed  Google Scholar 

  38. Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ (2005) GROMACS: fast, flexible, and free. J Comput Chem 26:1701–1718

    Article  Google Scholar 

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Acknowledgements

The present study was funded by Ferdowsi University of Mashhad of I.R.I with Grant No. 45321.

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Correspondence to Mohammad Hadi Sekhavati.

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Pirkhezranian, Z., Tanhaeian, A., Mirzaii, M. et al. Expression of Enterocin-P in HEK Platform: Evaluation of Its Cytotoxic Effects on Cancer Cell Lines and Its Potency to Interact with Cell-Surface Glycosaminoglycan by Molecular Modeling. Int J Pept Res Ther 26, 1503–1512 (2020). https://doi.org/10.1007/s10989-019-09956-7

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Keywords

  • Antimicrobial peptides
  • Entrocin-P
  • HEK expression system
  • Anticancer
  • Glycosaminoglycan
  • Structural bioinformatics