Evaluating cytotoxic effects of recombinant fragaceatoxin C pore forming toxin against AML cell lines

Document Type: Original Article

Authors

1 Protein Engineering Laboratory, Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

2 Department of Advanced Medical Sciences and Technologies, Faculty of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

3 Department of Biochemistry, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

4 Department of Microbiology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Abstract

Objective(s): Current therapeutic strategies for cancer are associated with side effects and lack of specificity in treatments. Biological therapies including monoclonal antibodies and immune effectors have been the subject of multiple research projects. Pore-forming proteins may become the other biological strategy to overcome the problems associated with current treatments. But detailed mechanisms of their action on target membranes remained to be elucidated. We aimed to study the cytotoxic effects of recombinant form of fragaceatoxin C on AML cell lines HL-60 and KG-1.
Materials and Methods: We cloned the FraC gene in pET-28a (+) bacterial expression vector and the expressed recombinant FraC protein was purified by affinity chromatography. Then, cytotoxic effects of the recombinant protein were examined on two AML cell lines, HL-60 and KG-1. Effects of serum and calcium ion were explored by hemolysis assay in more details.
Results: Our results showed that the recombinant C-terminal polyhistidine-tagged FraC protein has potent cytotoxic effects on both AML cell lines, with IC50=5.6, and 4.6 µg.ml-1 for HL-60 and KG-1 cells, respectively. Serum showed dose-dependent and also time-dependent inhibitory effects on the hemolytic and cytotoxic activities of the FraC protein. Pre-incubation of the toxin with different concentrations of calcium ion also inhibited hemolytic activity of FraC toxin.
Conclusion: Results of the present study showed that FraC has potential anti-tumor effects. By detailed investigation of the inhibition mechanism of serum and calcium effects in the future, it can be possible to design target sites for clinical applications of the toxin.

Keywords

Main Subjects


1. Döhner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med 2015; 373:1136-1152.
2. Seval GC, Ozcan M. Treatment of Acute Myeloid Leukemia in Adolescent and Young Adult Patients. J Clin Med 2015; 4:441-459.
3. Lin TL, Levy MY. Acute myeloid leukemia: focus on novel therapeutic strategies. Clin Med Insights Oncol 2012; 6:205-217.
4. Lynn RC, Poussin M, Kalota A, Feng Y, Low PS, Dimitrov DS, et al. Targeting of folate receptor beta on acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells. Blood 2015; 125:3466-3476.
5. Tettamanti S, Magnani CF, Biondi A, Biagi E. Acute myeloid leukemia and novel biological treatments: monoclonal antibodies and cell-based gene-modified immune effectors. Immunol Lett 2013; 155:43-46.
6. Horita H, Frankel AE, Thorburn A. Acute myeloid leukemia-targeted toxin activates both apoptotic and necroptotic death mechanisms. PLoS One 2008; 3:e3909.
7. Black J, McCubrey JA, Willingham M, Ramage J, Hogge D, Frankel A. Diphtheria toxin-interleukin-3 fusion protein (DT388IL3) prolongs disease-free survival of leukemic immunocompromised mice. Leukemia 2003; 17:155-159.
8. Alouf J. Pore-forming bacterial protein toxins: an overview.  Pore-forming toxins: Springer; 2001. p. 1-14.
9. Gouaux E. Channel-forming toxins: tales of transformation. Curr Opin Struct Biol 1997; 7:566-573.
10. Parker MW, Feil SC. Pore-forming protein toxins: from structure to function. Prog Biophys Mol Biol 2005; 88:91-142.
11. Gilbert R. Pore-forming toxins. Cell Mol Life Sci 2002; 59:832-844.
12. Gonzalez M, Bischofberger M, Pernot L, Van Der Goot F, Freche B. Bacterial pore-forming toxins: the (w) hole story? Cell Mol Life Sci 2008; 65:493-507.
13. Mechaly AE, Bellomio A, Gil-Cartón D, Morante K, Valle M, González-Mañas JM, et al. Structural insights into the oligomerization and architecture of eukaryotic membrane pore-forming toxins. Structure 2011; 19:181-191.
14. Rojko N, Dalla Serra M, Maček P, Anderluh G. Pore formation by actinoporins, cytolysins from sea anemones. Biochim Biophys Acta 2016;1858:446-456.
15. Tanaka K, Caaveiro JM, Morante K, González-Mañas JM, Tsumoto K. Structural basis for self-assembly of a cytolytic pore lined by protein and lipid. Nat Commun 2015; 6.
16. Ravindran VS, Kannan L, Venkateshvaran K. Biological activity of sea anemone proteins: II. Cytolysis and cell line toxicity. Indian J Exp Biol 2010; 48:1233-1236.
17. Jiang X, Chen H, Yang W, Liu Y, Liu W, Wei J, et al. Functional expression and characterization of an acidic actinoporin from sea anemone Sagartia rosea. Biochem Biophys Res Commun 2003; 312:562-570.
18. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual: Cold Spring Harbor Laboratory Cold Spring Harbor, NY; 1982.
19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-685.
20. Dal Peraro M, van der Goot FG. Pore-forming toxins: ancient, but never really out of fashion. Nat Rev Microbiol 2016; 14:77-92.
21. Mueller M, Grauschopf U, Maier T, Glockshuber R, Ban N. The structure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism. Nature 2009; 459:726-730.
22. Tweten RK. Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins. Infect Immun 2005; 73:6199-6209.
23. Tejuca M, Diaz I, Figueredo R, Roque L, Pazos F, Martinez D, et al. Construction of an immunotoxin with the pore forming protein StI and ior C5, a monoclonal antibody against a colon cancer cell line. Int Immunopharmacol 2004; 4:731-744.
24. Potrich C, Tomazzolli R, Dalla Serra M, Anderluh G, Malovrh P, Macek P, et al. Cytotoxic activity of a tumor protease-activated pore-forming toxin. Bioconjug Chem 2005; 16:369-376.
25. Panchal R, Smart M, Bowser D, Williams D, Petrou S. Pore-forming proteins and their application in biotechnology. Curr Pharm Biotechnol 2002; 3:99-115.
26. Yang WS, Park SO, Yoon AR, Yoo JY, Kim MK, Yun CO, et al. Suicide cancer gene therapy using pore-forming toxin, streptolysin O. Mol Cancer Ther 2006; 5:1610-1619.
27. Mechaly AE, Bellomio A, Morante K, Gonzalez-Manas JM, Guerin DM. Crystallization and preliminary crystallographic analysis of fragaceatoxin C, a pore-forming toxin from the sea anemone Actinia fragacea. Acta Crystallogr Sect F Struct Biol Cryst Commun 2009; 65:357-360.
28. Bellomio A, Morante K, Barlic A, Gutierrez-Aguirre I, Viguera AR, Gonzalez-Manas JM. Purification, cloning and characterization of fragaceatoxin C, a novel actinoporin from the sea anemone Actinia fragacea. Toxicon 2009; 54:869-880.
29. Imani M, Zarei Jaliani H, Kheirandish MH, Azadpour M. Recombinant production and affinity purification of the FraC pore forming toxin using hexa-His tag and pET expression cassette. Iranian J Basic Med Sci 2017; 20:380-385.
30. Anderluh G, Pungercar J, Krizaj I, Strukelj B, Gubensek F, Macek P. N-terminal truncation mutagenesis of equinatoxin II, a pore-forming protein from the sea anemone Actinia equina. Protein Eng 1997; 10:751-755.
31. Kristan K, Podlesek Z, Hojnik V, Gutierrez-Aguirre I, Guncar G, Turk D, et al. Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich. J Biol Chem 2004; 279:46509-46517.
32. Kheirandish MH, Zarei Jaliani H, Rahmani B. Application of a Seamless and Restriction Endonuclease-free Cloning Method to Produce Recombinant Full-length N-terminal His-tagged Streptolysin O in E. coli. Int J Medl Lab 2017; 4:189-200.
33. Fedorov S, Dyshlovoy S, Monastyrnaya M, Shubina L, Leychenko E, Kozlovskaya E, et al. The anticancer effects of actinoporin RTX-A from the sea anemone Heteractis crispa (=Radianthus macrodactylus). Toxicon 2010; 55:811-817.