Development of the therapeutic regimen based on the synergistic activity of cyclophosphamide and double-stranded DNA preparation which results in complete cure of mice engrafted with Krebs-2 ascites

Cumulative evidence obtained in this series of studies has guided the logic behind the development of a novel composite dsDNA-based preparation whose therapeutic application according to the specific regimen completely cures the mice engrafted with otherwise lethal Krebs-2 ascites. The likely mechanism involves elimination of TAMRA+ tumor-inducing stem cells (TISCs) from Krebs-2 tumors. We performed quantitative analysis of TISC dynamics in Krebs-2  ascites following treatment with the cytostatic drug cyclophosphamide (CP) and untreated control cells. In intact ascites, TISC percentage oscillates around a certain value. Following CP treatment and massive apoptosis of committed cancer cell subpopulation, we observed relative increase in TISC percentage, which is consistent with reduced susceptibility of TISCs to CP. Nonetheless, this treatment apparently synchronizes TISCs in a cell cycle phase when they become sensitive to further drug treatments. We describe the regimen of synergistic DNA + CP activity against Krebs-2 ascites. This protocol results in a complete cure of 50 % of Krebs-2 engrafted mice and involves three metronomic injections of CP exactly at the timepoints when repair cycles are about to finish combined with dsDNA injections 18 hours following each CP injection. The “final shot” uses CP + DNA treatment, which targets the surviving yet highly synchronized and therefore treatmentsensitive cells. The first three CP/DNA injections appear to arrest Krebs-2 cells in late S-G2-M phase and result in their simultaneous progression into G1-S phase. The timing of the “final shot” is crucial for the successful treatment, which eradicates tumorigenic cell subpopulation from Krebs-2 ascites. Additionally, we quantified the changes in several biochemical, cellular and morphopathological parameters in mice throughout different treatment stages.

1. Alyamkina E.A., Nikolin V.P., Popova N.A., Minkevich A.M., Kozel A.V., Dolgova E.V., Efremov Y.R., Bayborodin S.I., Andrushkevich O.M., Taranov O.S., Omigov V.V., Rogachev V.A., Proskurina A.S., Vereschagin E.I., Kiseleva E.V., Zhukova M.V., Ostanin A.A., Chernykh E.R., Bogachev S.S., Shurdov M.A. Combination of cyclophosphamide and double-stranded DNA demonstrates synergistic toxicity against established xenografts. Cancer Cell Int. 2015;15(32). DOI 10.1186/s12935-015-0180-6.

2. Deleyrolle L.P., Ericksson G., Morrison B.J., Lopez J.A., Burrage K., Burrage P., Vescovi A., Rietze R.L., Reynolds B.A. Determination of somatic and cancer stem cell self-renewing symmetric division rate using sphere assays. PLoS One. 2011;6(1):e15844. DOI 10.1371/journal.pone.0015844.

3. Dolgova E.V., Alyamkina E.A., Efremov Y.R., Nikolin V.P., Popova N.A., Tyrinova T.V., Kozel A.V., Minkevich A.M., Andrushkevich O.M., Zavyalov E.L., Romaschenko A.V., Bayborodin S.I., Taranov O.S., Omigov V.V., Shevela E.Y., Stupak V.V., Mishinov S.V., Rogachev V.A., Proskurina A.S., Mayorov V.I., Shurdov M.A., Ostanin A.A., Chernykh E.R., Bogachev S.S. Identification of cancer stem cells and a strategy for their elimination. Cancer Biol. Ther. 2014;15(10):1378- 1394. DOI 10.4161/cbt.29854.

4. Dolgova E.V., Likhacheva A.S., Orischenko K.E., Alyamkina E.A., Bogachev S.S., Shurdov M.A. Repair of interstrand crosslinks in a DNA molecule. Informatsionnyy vestnik VOGiS = The Herald of Vavilov Society for Geneticists and Breeders. 2010;14(2):332-356. (in Russian)

5. Dolgova E.V., Potter E.A., Proskurina A.S., Minkevich A.M., Chernych E.R., Ostanin A.A., Efremov Y.R., Nikolin V.P., Popova N.A., Kolchanov N.A., Bogachev S.S. Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line. Stem Cell Res. Ther. 2016;7(1):76. DOI 10.1186/s13287-016-0338-8.

6. Gómez-López S., Lerner R.G., Petritsch C. Asymmetric cell division of stem and progenitor cells during homeostasis and cancer. Cell. Mol. Life Sci. 2014;71:575-597. DOI 10.1007/s00018-013-1386-1.

7. Lathia J.D., Hitomi M., Gallagher J., Gadani S.P., Adkins J., Vasanji A., Liu L., Eyler C.E., Heddleston J.M., Wu Q., Minhas S., Soeda A., Hoeppner D.J., Ravin R., McKay R.D., McLendon R.E., Corbeil D., Chenn A., Hjelmeland A.B., Park D.M., Rich J.N. Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions. Cell Death Disease. 2011;2(9). DOI 10.1038/cddis.2011.80.

8. Peshkova I.V., Bogachev S.S., Drobyazgin E.A., Chikinev Y.V., Mitrofanov I.M., Piontkovskaya K.A., Vereschagin E.I. Effect of glutamine on the nucleic acids exchange during the perioperative period in reconstructive esophagus surgery. J. Nutr. Health Food. 2015;2(4). DOI 10.15406/jnhfe.2015.02.00058.

9. Pine S.R., Liu W. Asymmetric cell division and template DNA cosegregation in cancer stem cells. Front Oncol. 2014;4(226). DOI 10.3389/fonc.2014.00226.

10. Potter Е.А., Dolgova Е.V., Minkevich А.М., Efremov Y.R., Taranov О.S., Omigov V.V., Nikolin V.P., Popova N.A., Proskurina А.S., Vereschagin Е.I., Kozel А.V., Rogachev V.А., Petrov D.B., Ostanin А.А., Chernykh Е.R., Kolchanov N.А., Bogachev S.S. Therapeutic effects of cyclophosphamide, dsDNA preparations and their combinations against Krebs-2 ascites cancer cells and various cancer transplants. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2016a;20(1):96-107. DOI 10.18699/VJ15.116. (in Russian)

11. Potter Е.А., Dolgova Е.V., Minkevich А.М., Nikolin V.P., Popova N.A., Efremov Y.R., Baiborodin S.I., Rogachev V.А., Proskurina А.S., Kozel А.V., Taranov О.S., Omigov V.V., Vereschagin Е.I., Petrov D.B., Ostanin А.А., Chernykh Е.R., Kolchanov N.А., Bogachev S.S. Analysis of different therapeutic schemes combining cyclophosphamide and double-stranded DNA preparations for eradication of Krebs-2 primary ascites in mice. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2016b;20(1):108-124. DOI 10.18699/VJ15.117. (in Russian)

12. Potter E.A., Dolgova E.V., Minkevich A.M., Nikolin V.P., Popova N.A., Efremov Ya.R., Baiborodin S.I., Rogachev V.A., Proskurina A.S., Kozel A.V., Taranov O.S., Omigov V.V., Vereschagin E.I., Ostanin А.А., Chernykh Е.R., Kolchanov N.А., Bogachev S.S. Eradication of Krebs-2 primary ascites via a single-injection regimen of double-stranded DNA after each cyclophosphamide injection. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2016с;20(5):716-722. DOI 10.18699/VJ16.161. (in Russian)

13. Potter E.A., Dolgova E.V., Proskurina A.S., Minkevich A.M., Efremov Ya.R., Taranov O.S., Omigov V.V., Nikolin V.P., Popova N.A., Bayborodin S.I., Ostanin A.A., Chernykh E.R., Kolchanov N.A., Shurdov M.A., Bogachev S.S. A strategy to eradicate well-developed Krebs- 2 ascites in mice. Oncotarget. 2016;7(10):11580-11594. DOI 10.18632/oncotarget.7311.