Celecoxib and radioresistant glioblastoma-derived CD133+ cells: improvement in radiotherapeutic effects

Laboratory investigation

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Glioblastoma, the most common primary brain tumor, has a poor prognosis, even with aggressive resection and chemoradiotherapy. Recent studies indicate that CD133+ cells play a key role in radioresistance and recurrence of glioblastoma. Cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandins, is over-expressed in a variety of tumors, including CD133+ glioblastomas. The COX-2–derived prostaglandins promote neovascularization during tumor development, and conventional radiotherapy increases the proportion of CD133+ cells rather than eradicating them. The aim of the present study was to investigate the role of celecoxib, a selective COX-2 inhibitor, in enhancing the therapeutic effects of radiation on CD133+ glioblastomas.


Cells positive for CD133 were isolated from glioblastoma specimens and characterized by flow cytometry, then treated with celecoxib and/or ionizing radiation (IR). Clonogenic assay, cell irradiation, cell cycle analysis, Western blot, and xenotransplantation were used to assess the effects of celecoxib alone, IR alone, and IR with celecoxib on CD133+ and CD133 glioblastoma cells. Three separate xenotransplantation experiments were carried out using 310 severe combined immunodeficient (SCID) mice: 1) an initial tumorigenicity evaluation in which 3 different quantities of untreated CD133 cells or untreated or pretreated CD133+ cells (5 treatment conditions) from 7 different tumors were injected into the striatum of 2 mice (210 mice total); 2) a tumor growth study (50 mice); and 3) a survival study (50 mice). For these last 2 studies the same 5 categories of cells were used as in the tumorigenicity (untreated CD133 cells, untreated or pretreated CD133+ cells, with pretreatment consisting of celecoxib alone, IR alone, or IR and celecoxib), but only 1 cell source (Case 2) and quantity (5 × 104 cells) were used.


High levels of COX-2 protein were detected in the CD133+ but not the CD133 glioblastoma cells. The authors further demonstrated that 30 μM celecoxib was able to effectively enhance the IR effect in inhibiting colony formation and increasing IR-mediated apoptosis in celecoxib-treated CD133+ glioblastoma cells. Furthermore, reduction in radioresistance was correlated with the induction of G2/M arrest, which was partially mediated through the increase in the level of phosphorylated-cdc2. In vivo xenotransplant analysis further confirmed that CD133+-associated tumorigenicity was significantly suppressed by celecoxib treatment. Importantly, pretreatment of CD133+ glioblastoma cells with a combination of celecoxib and IR before injection into the striatum of SCID mice resulted in a statistically significant reduction in tumor growth and a statistically significant increase in the mean survival rate of the mice.


Celecoxib combined with radiation plays a critical role in the suppression of growth of CD133+ glioblastoma stemlike cells. Celecoxib is therefore a radiosensitizing drug for clinical application in glioblastoma.

Abbreviations used in this paper:bFGF = basic fibroblast growth factor; COX = cyclooxygenase; CSC = cancer stem cell; EGF = epidermal growth factor; GSC = glioblastoma stemlike cell; IC50 = half maximal inhibitory concentration; IR = ionizing radiation; NSCLC = non–small cell lung cancer; PBS = phosphate-buffered saline; PG = prostaglandin; PI = propidium iodide; RT-PCR = reverse transcriptase polymerase chain reaction; SB = spheroid body; SCID = severe combined immunodeficient; SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling.

Article Information

* Drs. Chiou, Hueng, Tai, and Huang contributed equally to this article.

Address correspondence to: Hsin-I Ma, M.D., Ph.D., Department of Neurological Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan. email: uf004693@mail2000.com.tw.

Please include this information when citing this paper: published online November 5, 2010; DOI: 10.3171/2009.11.JNS091396.

© AANS, except where prohibited by US copyright law.



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    Isolation and characterization of CD133+ cells from glioblastoma specimens from Case 2 (A–C) and Cases 1–3 (D). A: Scatterplots showing the results of flow cytometry. After cells were isolated with magnetic bead sorting, the percentages of CD133+ and CD133 cells isolated from glioblastoma samples were measured by flow cytometry. B: Evaluation of the ability of CD133+ and CD133 glioblastoma cells to form SBs in vitro (in serum-free medium with bFGF and EGF). C: Comparison of the COX-2 mRNA expression levels in CD133+ and CD133 glioblastoma cells from 7 patients; results of quantitative RT-PCR. D: Expression of COX-2 protein in glioblastoma-CD133+/– was measured by Western blot analysis. Pt = Patient. Data shown in B and C are the mean values (± SDs) of 3 experiments. *p < 0.001.

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    Radioresistance, asymmetrical self-renewal division, and sphere formation in CD133+ glioma stemlike cells (1XM cells) from Case 2. A and B: Flow cytometry results and bar graphs showing the composition of the CD133-deficient population (0XM cells; low CD133+) and glioblastoma-CD133+ GSCs (1XM cells; high CD133+). The x axis values in panel A represent fluorescence intensity (count refers to number of cells). Data are from triplicate samples. C: Photomicrographs. The 1XM (but not 0XM) cells formed primary neurospheres (1° sphere) and secondary neurospheres (2° sphere) in stem cell culture medium. Original magnification × 100. D: Photomicrographs. The 1XM cells (high CD133+) expressed typical stem cell markers (CD133 and nestin). Original magnifications × 200 (CD133) and × 400 (nestin). E: Representative photographs of 0XM and 1XM colony formation (in 10-cm dishes) 12 days after irradiation with 5 Gy and bar graph showing significant differences in colony formation. The bars represent the means of 3 experiments. The medium (DMEM with 10% fetal bovine serum) was changed twice a week. Colonies were stained with methylene blue.

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    Inhibition of cell growth and increase in radiosensitivity of CD133+ glioblastoma cells (1XM cells) with celecoxib treatment. Data shown are the means ± SDs of 3 experiments; cells were obtained from Case 2. A: Bar graph showing a significant difference in 1XM (high CD133+) colony formation 4 days following treatment with celecoxib (Celebrex 0, 10, 20, and 30 μM), as compared with treatment with the indicated celecoxib dose and 5 Gy IR. B: Representative photographs of petri dishes containing 1XM (high CD133+) clones that developed after cells were treated with ethanol (control), ethanol and IR (5 Gy), 30 μM celecoxib (Cx), or both celecoxib and IR (5 Gy). C: Line graph showing the IC50 of celecoxib based on 4-day treatment. D: Bar graph showing the percentages of cells that were apoptotic as determined by TUNEL assay. *p < 0.0005.

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    The effect of celecoxib and IR on the cell cycle of CD133+ GSCs. Data shown are the means ± SDs of 3 experiments; cells were obtained from Case 2. A: After 1XM cells (high CD133+) were exposed to 0 or 30 μM celecoxib overnight, they were stained with PI and analyzed by flow cytometry to determine the percentages of sub-G1 and G2/M cells. Celecoxib significantly increased the sub-G1 fraction at 7 days after treatment but had less effect on the G2/M fraction. B: Samples of 0XM (low CD133+) and 1XM (high CD133+) cells exposed to 30 μM celecoxib, 5 Gy IR, and 30 μM celecoxib plus 5 Gy IR were stained with PI and analyzed by flow cytometry.

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    The effect of celecoxib combined with IR on phosphorylated-cdc2 levels in treated CD133+ glioblastoma cells. Data shown are the means ± SDs of 3 experiments; cells were obtained from Case 2. A: Western blot and bar graph. Extracts of 0XM cells and 1XM cells treated with 5 Gy IR and/or 30 μM celecoxib were subjected to Western blot analysis with antibodies to cdc2, phosphorylated-cdc2 (pCDC2), COX-2, and beta actin (loading control). Representative example from 1 of 3 experiments. The fold change in levels of phosphorylated-cdc2 was calculated from 3 independent experiments. B: Cells were irradiated with 0, 2, 4, 6, 8, or 10 Gy, and the viable cells were counted after 24 hours. Cel-10 = 10 μM celecoxib; Cel-30 = 30 μM celecoxib. *p < 0.0005.

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    Evaluation of in vivo tumorigenicity and survival time in xenotransplanted animals (5 × 104 CD133+ cells per mouse). Cells were obtained from Case 2. A: In vivo tumor growth was assessed by histological examination. H & E. Bar = 200 μm. B: Tumor volumes in mice receiving cells treated with both celecoxib (Cel) and IR were significantly lower than in mice receiving CD133+ cells alone, celecoxib-treated CD133+ cells, or IR-treated CD133+ cells. C: Analysis showed that the mean survival rate of CD133+ cell–xenotransplanted SCID mice was significantly prolonged by pretreatment of the cells with celecoxib and IR. *p < 0.0005. GBM = glioblastoma.


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