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

Restricted access

Object

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).

Methods

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.

Results

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.

Conclusions

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: tjjiaqiang@yahoo.com.cn.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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).

  • View in gallery

    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.

  • View in gallery

    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.

References

  • 1

    Ayene ISFord LPKoch CJ: Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and gamma radiation. Mol Cancer Ther 4:5295362005

  • 2

    Furnari FBFenton TBachoo RMMukasa AStommel JMStegh A: Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev 21:268327102007

  • 3

    Gullo CAu MFeng GTeoh G: The biology of Ku and its potential oncogenic role in cancer. Biochim Biophys Acta 1765:2232342006

  • 4

    Guo DWang BHan FLei T: RNA interference therapy for glioblastoma. Expert Opin Biol Ther 10:9279362010

  • 5

    He FLi LKim DWen BDeng XGutin PH: Adenovirus-mediated expression of a dominant negative Ku70 fragment radiosensitizes human tumor cells under aerobic and hypoxic conditions. Cancer Res 67:6346422007

  • 6

    Henderson MAFakiris AJTimmerman RDWorth RMLo SSWitt TC: Gamma knife stereotactic radiosurgery for lowgrade astrocytomas. Stereotact Funct Neurosurg 87:1611672009

  • 7

    Heppner PASheehan JPSteiner LE: Gamma knife surgery for low-grade gliomas. Neurosurgery 62:Suppl 27557622008

  • 8

    Kida YYoshimoto MHasegawa T: Radiosurgery for intracranial gliomas. Prog Neurol Surg 22:1221282009

  • 9

    Komuro YWatanabe THosoi YMatsumoto YNakagawa KSaito S: Prediction of tumor radiosensitivity in rectal carcinoma based on p53 and Ku70 expression. J Exp Clin Cancer Res 22:2232282003

  • 10

    Kondziolka DLunsford LDLoeffler JSFriedman WA: Radiosurgery and radiotherapy: observations and clarifications. J Neurosurg 101:5855892004

  • 11

    Li GCHe FShao XUrano MShen LKim D: Adenovirus-mediated heat-activated antisense Ku70 expression radiosensitizes tumor cells in vitro and in vivo. Cancer Res 63:326832742003

  • 12

    Li GCOuyang HLi XNagasawa HLittle JBChen DJ: Ku70: a candidate tumor suppressor gene for murine T cell lymphoma. Mol Cell 2:181998

  • 13

    Li YJia QZhang JHan LXu DZhang A: Combination therapy with Gamma Knife radiosurgery and antisense EGFR for malignant glioma in vitro and orthotopic xenografts. Oncol Rep 23:158515912010

  • 14

    Louis DNOhgaki HWiestler ODCavenee WKBurger PCJouvet A: The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:971092007. (Erratum in Acta Neuropathol 114:547 2007)

  • 15

    Mahaney BLMeek KLees-Miller SP: Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. Biochem J 417:6396502009

  • 16

    Omori STakiguchi YSuda ASugimoto TMiyazawa HTakiguchi Y: Suppression of a DNA double-strand break repair gene, Ku70, increases radio- and chemosensitivity in a human lung carcinoma cell line. DNA Repair (Amst) 1:2993102002

  • 17

    Shrivastav MDe Haro LPNickoloff JA: Regulation of DNA double-strand break repair pathway choice. Cell Res 18:1341472008

  • 18

    Tuteja RTuteja N: Ku autoantigen: a multifunctional DNA-binding protein. Crit Rev Biochem Mol Biol 35:1332000

  • 19

    Ulm AJ IIIFriedman WABradshaw PFoote KDBova FJ: Radiosurgery in the treatment of malignant gliomas: the University of Florida experience. Neurosurgery 57:5125172005

  • 20

    Urano MHe FMinami ALing CCLi GC: Response to multiple radiation doses of human colorectal carcinoma cells infected with recombinant adenovirus containing dominantnegative Ku70 fragment. Int J Radiat Oncol Biol Phys 77:8778852010

  • 21

    Vandersickel VMancini MMarras EWillems PSlabbert JPhilippé J: Lentivirus-mediated RNA interference of Ku70 to enhance radiosensitivity of human mammary epithelial cells. Int J Radiat Biol 86:1141242010

  • 22

    Wang LWShiau CYChung WYWu HMGuo WYLiu KD: Gamma Knife surgery for low-grade astrocytomas: evaluation of long-term outcome based on a 10-year experience. J Neurosurg 105:Suppl1271322006

  • 23

    Xu DJia QLi YKang CPu P: Effects of Gamma Knife surgery on C6 glioma in combination with adenoviral p53 in vitro and in vivo. J Neurosurg 105:Suppl2082132006

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 99 99 37
Full Text Views 72 72 0
PDF Downloads 32 32 1
EPUB Downloads 0 0 0

PubMed

Google Scholar