Article Figures & Data
Figures
- Figure 1
The basic Karanahan approach parameters and treatment schedule. (A) TAMRA+ cells in in vitro U87 cell culture. (B) Cross-examination of U87 cells with regard to their ability both to bind antibodies to the surface marker CD133 and to internalize the TAMRA-DNA probe. Arrows indicate TAMRA+ cells, arrowheads indicate CD133+ cells. (C) Combined fluorescence profile along the vector crossing the single U87 cell [result of processing the image obtained with LSM 780 NLO confocal microscope (Zeiss) in Zen software package]. Arrow indicates the site of colocalization of TAMRA-DNA and anti-CD133 antibodies signals. (D) FACS assay of the content of TAMRA+/CD133+ cells in in vitro U87 cell culture. DAPI, chromatin staining; TAMRA, fluorescence of the TAMRA-labeled DNA probe; APC, anti-CD133 antibodies. (E) Profile of DNA DSB occurrence in U87 cells exposed to MMC. The number of DSBs was determined as the “tail moment” during the comet assay. The average value ± 99% confidence intervals are given; confidence was estimated relative to the “zero point” using the Student’s criterion in the Statistica 10 software package, **P < 0.01, #P < 0.001. (F) Schedule for MMC treatments (three rounds every 36 h) as well as for the cell cycle assays.
- Figure 2
Basic Karanahan approach parameters and treatment schedule. (A) Results of the cell cycle assays in U87 cells prior to the treatment with MMC, 18, 54 and 90 h later as well as on days 6–9 after the first treatment. G1, S, and G2/M, phases of the cell cycle; subG1, apoptotic (presumably) cells. (B) Principle therapeutic scheme for treating U87 glioblastoma cells in vitro. Arrows indicate the appropriate timings for administration of cytostatic agent and composite DNAmix preparation. The dashed line displays the concomitant number of DSBs induced by the exposure of U87 cells to a cytostatic agent. (C) Percentage of TAMRA+ cells in U87 cell culture after treatments with MMC and DNAmix preparation according to the Karanahan approach. The average value ± 99% CI are given; confidence was estimated relative to the control using the Mann–Whitney criterion in the Statistica 10 software package, *P < 0.05. (D) Cytological preparations of U87 cells before (control) and on days 3, 7, and 15 since the start of therapy. The figure represents the merged image of DAPI (chromatin) and TAMRA (exogenous DNA) staining. For the control and MMC + DNAmix groups, images of cells in transmitting light are also given. MMC, group treated with cytostatic only; MMC + DNAmix, group treated with cytostatic in combination with DNA-based preparation.
- Figure 3
Intracerebral grafting of pretreated in vitro U87 glioblastoma cells into SCID mice. (A) Treatment schematic. (B) NMR tomography of the brain of SCID mice on day 24 after intracerebral inoculation of U87 human glioblastoma cells. Control, mice inoculated with untreated U87 cells; DNA, mice inoculated with U87 cells pretreated with DNAmix only; MMC, mice inoculated with U87 cells pretreated with MMC only; MMC + DNAmix, mice inoculated with U87 cells pretreated with MMC in combination with the composite dsDNA preparation.
- Figure 4
Therapeutic treatment of experimental mice with developed subcutaneous xenografts of U87 human glioblastoma. (A) Graft growth rates in mice with an average initial tumor size of 15 mm3 (n = 7 per group). (B) Graft growth rates in mice with the average initial tumor size of 92 mm3 (n = 6 per group). The median ± standard error of the mean are given; confidence was estimated using the Mann–Whitney criterion in the Statistica 10 software package, *P < 0.05. (C) Treated animals. (D) Animals from the CP + DNAmix group with a characteristic symptom complex manifestation: 1) animal with severe anorexia; 2) animal with hind limb paralysis (indicated by the arrow).
- Figure 5
Magnetic resonance images (MRIs) of tumor nodes and survival rates of mice. (A) Schematic Karanahan approach with the schedule and dosages of CP and DNAmix administration. (B) Pictures of four sequential MRIs taken during the experiment. Preoperative MRIs indicate the presence of individual neoplastic formations with clear boundaries localized subcortically in the projection of parietal lobes (marked with white arrows) in all three groups. Postoperative MRIs indicate the total tumor resection (100% removal of tumor bulk) achieved in all the cases presented (areas of surgical intervention are marked with red arrows). On day 17 after the therapy is finished, MRIs indicate a dissemination of tumors through the cerebrospinal liquor paths to the brain basal zones (encircled with white dotted lines) in the CP and control groups. Thirty days since the end of therapy, a noticeable increase in tumor volume associated with infiltrative growth was observed in the CP and control groups, whereas the CP + DNAmix group displayed no signs of relapse. (C) Mice survival rates (Kaplan–Meier curves) during the experiment. Preoperative MRI is considered the start of the experiment. (D) Growth rates of tumors in individual animals by the experimental groups: control, CP, and CP + DNAmix. (E) Histological examination of the relapsing tumor from a mouse in the CP + DNAmix group.
Tables
Group Animal number Preoperative tumor volumetry (mm3) Postoperative tumor volumetry (mm3) Radicality of tumor resection (%) Cyclophosphamide + DNAmix 1_1 4.692 0 100* 1_2 3.103 0 100* 1_3 5.622 2.222 61† 1_4 2.446 0 100* Cyclophosphamide 2_1 5.662 0 100* 2_2 2.818 2.718 – 2_3 1.974 0 100* 2_4 63.364 16.251 75† Control 3_1 3.434 0 100* 3_2 41.736 16.548 61† 3_3 0.134 0.239 – 3_5 5.106 0 100* *Total resection. †Partial resection. BBB, blood–brain barrier; CNS, central nervous system; CP, cyclophosphamide; CSC, cancer stem cell; DC, dendritic cell; dsDNA, double-stranded DNA; HR, homologous repair; ICL, interstrand cross-link; MDSC, myeloid-derived suppressor cell; MMC, mitomycin C; MOF, multiple organ failure; MTIC, monomethyl triazen imidazole carboxamide; NER, nucleotide excision repair; TM, tail moment; TMZ, temozolomide.
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