Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

Laboratory investigation

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The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT).


First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses.


The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged.


Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Abbreviations used in this paper: DNA-PKcs = DNA-dependent protein kinase catalytic subunit; DSB = double-strand break; FCS = fetal calf serum; GKT = Gamma Knife treatment; MMP = matrix metalloproteinase; MTT = 3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; NHEJ = nonhomologous end-joining; PCNA = proliferating cell nuclear antigen; siRNA = small interfering RNA; TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling.

Article Information

Drs. Jia and Yanhe Li contributed equally to this work.

Address correspondence to: Desheng Xu, M.D., Department of Neurosurgery and Gamma Knife Center, Tianjin Medical University 2nd Hospital, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China. email:

© AANS, except where prohibited by US copyright law.



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    Western blots (upper) and bar graph (lower) showing that the expression of Ku70 protein inhibited by Ad-Ku70shRNA in C6 cells was significantly decreased compared with the expression of Ku70 protein in the Control and Scramble groups. Black triangle indicates p < 0.01 compared with the Control and Scramble groups.

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    Line graph showing the results of the MTT assays. Treated and control C6 glioma cells (4 × 103 per well) were plated into a 96-well plate and tested every 24 hours. The y axis indicates the comparative survival rate of glioma cells in the Control, Scramble, Ad-Ku70shRNA, GKT, and Combined Treatment groups. The x axis represents days after cell implantation in the 96-well plate. One-way ANOVA was performed. Black triangle indicates p < 0.01 compared with the Control group; black triangle plus asterisk indicates p < 0.01 compared with the GKT group.

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    Expression of MMP-2 and MMP-9 in the Control and treated groups. Upper: Western blot showing that expression of MMP-2 and MMP-9 was decreased in both the GKT and Combined Treatment groups. Lower: Bar graph showing relative protein levels of MMP-2 and MMP-9, as normalized to the β-actin level. Black triangle indicates p < 0.01 compared with the Control group; black triangle plus asterisk indicates p < 0.01 compared with the GKT group.

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    Cell cycle and apoptosis evaluated by flow cytometry. A: Cell cycle profiles after propidium iodide staining. Both GKT and combined treatment induced G1 arrest in C6 glioma cells. B: Graphic representation of the cell cycle profiles shown in A. C: Analysis of early apoptosis using Annexin V staining. Both GKT and combined treatment led to C6 glioma cell apoptosis. D: Graphic representation of the data shown in C. One-way ANOVA was performed. Black triangle indicates p < 0.01 (compared with control group). Black triangle plus asterisk indicates p < 0.01 (compared with GKT group).

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    Photomicrographs showing the results of immunohistochemical analyses of C6 xenograft tumors. Both PCNA and MMP-9 expression were decreased in the GKT and Combined Treatment groups, whereas Caspase 3 and the apoptosis index (TUNEL) were increased simultaneously. Bar = 10 μm.

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    Graph showing the results of the survival analysis of rats harboring intracerebral C6 gliomas with and without shRNA transduction and exposure to GKT. 1 = Control group; 2 = Scramble group; 3 = Ad-Ku70shRNA group; 4 = GKT group; 5 = Combined Treatment group.


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