ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
"Biotechnologia Acta" V. 11, No 2, 2018
Р. 64-71, Bibliography 34, English
Universal Decimal Classification: 616.931:616-006.6
https://doi.org/10.15407/biotech11.02.064
K. Y. Manoilov, O. I. Krynina, A. Ju. Labyntsev, S. I. Romaniuk, D. V. Kolybo
Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv
The aim of the work was to evaluate in vitro the cytostatic effect of recombinant fragments of the non-toxic point mutant of diphtheria toxin — CRM197, which was suggested as a potent medication for treatment of triple negative breast cancer. For this purpose, non-toxic recombinant derivatives of diphtheria toxin — CRM197, subunit B SbB and receptor domain Rd had been isolated by Ni-NTA agarose affinity chromatography and their effect on the growth of individual colonies of triple negative breast cancer MDA–MB–231 cells were characterized by such parameters as average colony area, perimeter and circularity index. According to the obtained results, CRM197 and SbB, whose molecules contain the translocation domain Td, exhibited the same cytostatic effect against MDA–MB–231 cells, reducing the area and perimeter of individual colonies. Rd protein did not affect the last two parameters that characterize the size of the colonies, but changed the form of the margin of colonies, as evidenced by an increase in the circularity index.
It is supposed that Td may be involved in the implementation of cytostatic action due to its inherent pore-forming activity in relation to lipid membranes. It is concluded that Rd and Td, unlike the catalytic domain of diphtheria toxin, play important roles in the implementation of the cytotoxic properties of CRM197, while SbB consisting of Rd and Td is the structural DT fragment of smallest molecular weight that can be used as the analog of CRM197.
Key words: CRM197, diphtheria toxin, HB-EGF, toxoid, triple negative breast cancer.
© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2018
References
1. Uchida T., Pappenheimer A. M., Greany R. Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J. Biol. Chem. 1973, 248 (11), 3838–3844.
2. Giannini G., Rappuoli R., Ratti G. The aminoacid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Res. 1984, 12 (10), 4063–4069. https://doi.org/10.1093/nar/12.10.4063
3. Buzzi S., Maistrello I. Inhibition of growth of Erlich tumors in Swiss mice by diphtheria toxin. Cancer Res. 1973, 33 (10), 2349–2353.
4. Tang X.-H., Deng S., Li M., Lu M.-S. Crossreacting material 197 reverses the resistance to paclitaxel in paclitaxel-resistant human ovarian cancer. Tumour Biol. 2016, 37 (4), 5521–5528. https://doi.org/10.1007/s13277-015-4412-0
5. Yagi H., Yotsumoto F., Sonoda K., Kuroki M., Mekada E., Miyamoto S. Synergistic antitumor effect of paclitaxel with CRM197, an inhibitor of HB-EGF, in ovarian cancer. Int. J. Cancer. 2009, 124 (6), 1429–1439. https://doi.org/10.1002/ijc.24031
6. Nam S. O., Yotsumoto F., Miyata K., Fukagawa S., Odawara T., Manabe S., Ishikawa T., Kuroki M., Yasunaga S., Miyamoto S. Anti-tumor Effect of Intravenous Administration of CRM197 for Triple-negative Breast Cancer Therapy. Anticancer Res. 2016, 36 (7), 3651–3657.
7. Buzzi S., Rubboli D., Buzzi G., Buzzi A. M., Morisi C., Pironi F. CRM197 (nontoxic diphtheria toxin): effects on advanced cancer patients. Cancer Immunol. Immunother. CII. 2004, 53 (11), 1041–1048. https://doi.org/10.1007/s00262-004-0546-4
8. Nam S. O., Yotsumoto F., Miyata K., Suzaki Y., Yagi H., Odawara T., Manabe S., Ishikawa T., Kuroki M., Mekada E., Miyamoto S. Preclinical Study of BK-UM, a Novel Inhibitor of HB-EGF, for Ovarian Cancer Therapy. Anticancer Res. 2014, 34 (8), 4615–4620.
9. Tsujioka H., Fukami T., Yotsumoto F., Ueda T., Hikita S., Takahashi Y., Kondo H., Kuroki M., Miyamoto S. A possible clinical adaptation of CRM197 in combination with conventional chemotherapeutic agents for ovarian cancer. Anticancer Res. 2011, 31 (7), 2461–2465.
10. Lian C., Ruan L., Shang D., Wu Y., Lu Y., L? P., Yang Y., Wei Y., Dong X., Ren D., Chen K., Liu H., Tu Z. Heparin-Binding Epidermal Growth Factor-Like Growth Factor as a Potent Target for Breast Cancer Therapy. Cancer Biother. Radiopharm. 2016, 31 (3), 85–90. https://doi.org/10.1089/cbr.2015.1956
11. Yotsumoto F., Oki E., Tokunaga E., Maehara Y., Kuroki M., Miyamoto S. HB-EGF orchestrates the complex signals involved in triple-negative and trastuzumab-resistant breast cancer. Int. J. Cancer. 2010, 127 (11), 2707–2717. https://doi.org/10.1002/ijc.25472
12. Dateoka S., Ohnishi Y., Kakudo K. Effects of CRM197, a specific inhibitor of HB-EGF, in oral cancer. Med. Mol. Morphol. 2012, 45 (2), 91–97. https://doi.org/10.1007/s00795-011-0543-6
13. Sanui A., Yotsumoto F., Tsujioka H., Fukami T., Horiuchi S., Shirota K., Yoshizato T., Kawarabayashi T., Kuroki M., Miyamoto S. HB-EGF inhibition in combination with various anticancer agents enhances its antitumor effects in gastric cancer. Anticancer Res. 2010, 30 (8), 3143–3149.
14. Kunami N., Yotsumoto F., Ishitsuka K., Fu kami T., Odawara T., Manabe S., Ishikawa T., Tamura K., Kuroki M., Miyamoto S. Antitumor effects of CRM197, a specific inhibitor of HBEGF, in T-cell acute lymphoblastic leukemia. Anticancer Res. 2011, 31 (7), 2483–2488.
15. Mahamad P., Boonchird C., Panbangred W. High level accumulation of soluble diphtheria toxin mutant (CRM197) with co-expression of chaperones in recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 2016, 100 (14), 6319–6330. https://doi.org/10.1007/s00253-016-7453-4
16. Dukhovlinov I. V., Fedorova E. A., Bogomolova E. G., Dobrovolskaya O. A., Chernyaeva E. N., Al-Shekhadat R. I., Simbirtsev A. S. Production of recombinant protein CRM197 in Escherichia coli. Russ. J. Infect. Immun. 2015, 5 (1), 37. doi: 10.15789/2220-76192015-1-37-44.
17. Stefan A., Conti M., Rubboli D., Ravagli L., Presta E., Hochkoeppler A. Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli. J. Biotechnol. 2011, 156 (4), 245–252. https://doi.org/10.1016/j.jbiotec.2011.08.024
18. Wang F., Liu R., Lee S. W., Sloss C. M., Couget J., Cusack J. C. Heparin-binding EGF-like growth factor is an early response gene to chemotherapy and contributes to chemotherapy resistance. Oncogene. 2007, 26 (14), 2006–2016. doi: 10.1038/ sj.onc.1209999.
19. Zhou Z. N., Sharma V. P., Beaty B. T., RohJohnson M., Peterson E. A., Van Rooijen N., Kenny P. A., Wiley H. S., Condeelis J. S., Segall J. E. Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo. Oncogene. 2014, 33 (29), 3784–3793. https://doi.org/10.1038/onc.2013.363
20. Labyntsev A. J., Korotkevych N. V., Manoilov K. J., Kaberniuk A. A., Kolybo D. V., Komisarenko S. V. Recombinant fluorescent models for studying the diphtheria toxin. Russ. J. Bioorg. Chem. 2014, 40 (4), 401–409. doi: 10.1134/ S1068162014040086.
21. Kaberniuk A. A., Oliinyk O. S., Redchuk T. A., Romaniuk S. I., Kolybo D. V., Komisarenko S. V. Cloning of recombinant subunits of Corynebacterium diphtheriae diphtheria toxin and their expression in Escherichia coli. Dopov. Nats. akad. nauk Ukr. 2008, 3, 160–166. (In Ukrainian).
22. Kaberniuk A. A., Labyntsev A. I., Kolybo D. V., Oli?nyk O. S., Redchuk T. A., Korotkevych N. V., Horchev V. F., Karakhim S. O., Komisarenko S. V. Fluorescent derivatives of diphtheria toxin subunit B and their interaction with Vero cells. Ukr. Biokhim. Zh. 2009, 81 (1), 67–77. (In Ukrainian).
23. Labyntsev A. I., Korotkevich N. V., Kaberniuk A. A., Romaniuk S. I., Kolybo D. V., Komisarenko S. V. Interaction of diphtheria toxin B subunit with sensitive and insensitive mammalian cells. Ukr. Biokhim. Zh. 2010, 82 (6), 65–75. (In Ukrainian).
24. Labyntsev A. J., Kolybo D. V., Yurchenko E. S., Kaberniuk A. A., Korotkevych N. V., Komisarenko S. V. Effect of the T-domain on intracellular transport of diphtheria toxin. Ukr. Biochem. J. 2014, 86 (3), 77–87. https://doi.org/10.15407/ubj86.03.077
25. Labyntsev A. J., Korotkevych N. V., Kolybo D. V., Komisarenko S. V. Effect of diphtheria toxin T-domain on endosomal pH. Ukr. Biochem. J. 2015, 87 (4), 13–23. https://doi.org/10.15407/ubj87.04.013
26. Sch?gger H., von Jagow G. Tricinesodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987, 166 (2), 368–379. https://doi.org/10.1016/0003-2697(87)90587-2
27. Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., Tinevez J. Y., White D. J., Hartenstein V., Eliceiri K., Tomancak P., Cardona A. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 2012, 9 (7), 676–682. https://doi.org/10.1038/nmeth.2019
28. Rudolph R., Lilie H. In vitro folding of inclusion body proteins. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 1996, 10 (1), 49–56. https://doi.org/10.1096/fasebj.10.1.8566547
29. Korotkevych N. V., Labyntsev A. I., Kaberniuk A. A., Oli?nyk O. S., Romaniuk S. I., Kolybo D. V., Komisarenko S. V. Cytotoxicity of the B subunit of diphtheria toxin to human histocytic lymphoma U937. Ukr. Biokhim. Zh. 2009, 81 (4), 69–80. (In Ukrainian).
30. Donovan J. J., Simon M. I., Draper R. K., Montal M. Diphtheria toxin forms transmembrane channels in planar lipid bilayers. Proc. Natl. Acad. Sci. U. S. A. 1981, 78 (1), 172–176. https://doi.org/10.1073/pnas.78.1.172
31. Kagan B. L., Finkelstein A., Colombini M. Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes. Proc. Natl. Acad. Sci. U. S. A. 1981, 78 (8), 4950–4954. https://doi.org/10.1073/pnas.78.8.4950
32. Manoilov K. Y., Gorbatiuk O. B., Usenko M. O., Shatursky O. Y., Borisova T. O., Kolybo D. V. The characterization of purifed recombinant protein CRM197 as a tool to study diphtheria toxin. Dopov. Nats. acad. nauk Ukr. 2016, 9, 124–133. doi: 10.15407/ dopovidi2017.02.088. (In Ukrainian).
33. Manoilov K. Y., Gorbatiuk O. B., Usenko M. O., Shatursky O. Y., Borisova T. O., Kolybo D. V., Komisarenko S. V. The characterization of purified recombinant fragment B as a tool to study diphtheria toxin. Dopov. Nats. acad. nauk Ukr. 2017, 2, 88–99. doi: 10.15407/ dopovidi2016.09.124. (In Ukrainian).
34. Papini E., Sandon? D., Rappuoli R., Montecucco C. On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells. EMBO J. 1988, 7 (11), 3353–3359.