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

Laboratory investigation

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Object

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.

Methods

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.

Results

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.

Conclusions

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.

Headings

Figures

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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.

References

  • 1

    Altaner C: Glioblastoma and stem cells. Neoplasma 55:3693742008

  • 2

    Antonarakis ESHeath EIWalczak JRNelson WGFedor HDe Marzo AM: Phase II, randomized, placebo-controlled trial of neoadjuvant celecoxib in men with clinically localized prostate cancer: evaluation of drug-specific biomarkers. J Clin Oncol 27:498649932009

  • 3

    Bao SWu QMcLendon REHao YShi QHjelmeland AB: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444:7567602006

  • 4

    Beier DWischhusen JDietmaier WHau PProescholdt MBrawanski A: CD133 expression and cancer stem cells predict prognosis in high-grade oligodendroglial tumors. Brain Pathol 18:3703772008

  • 5

    Chang CJHsu CCYung MCChen KYTzao CWu WF: Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression. Biochem Biophys Res Commun 380:2362422009

  • 6

    Chen JCChen YSu YHTseng SH: Celecoxib increased expression of 14-3-3sigma and induced apoptosis of glioma cells. Anticancer Res 27:4B254725542007

  • 7

    Chiou SHKao CLChen YWChien CSHung SCLo JF: Identification of CD133-positive radioresistant cells in atypical teratoid/rhabdoid tumor. PLoS One 3:e20902008

  • 8

    Chiou SHKao CLLin HTTseng WSLiu RSChung CF: Monitoring the growth effect of xenotransplanted human medulloblastoma in an immunocompromised mouse model using in vitro and ex vivo green fluorescent protein imaging. Childs Nerv Syst 22:4754802006

  • 9

    Clement VSanchez Pde Tribolet NRadovanovic IRuiz i Altaba A: HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr Biol 17:1651722007

  • 10

    Debucquoy ARoels SGoethals LLibbrecht LVan Cutsem EGeboes K: Double blind randomized phase II study with radiation+5-fluorouracil±celecoxib for resectable rectal cancer. Radiother Oncol 93:2732782009

  • 11

    Dehdashti ARHegi MERegli LPica AStupp R: New trends in the medical management of glioblastoma multiforme: the role of temozolomide chemotherapy. Neurosurg Focus 20:4E62006

  • 12

    Furuta YHall ERSanduja SBarkley T JrMilas L: Prostaglandin production by murine tumors as a predictor for therapeutic response to indomethacin. Cancer Res 48:300230071988

  • 13

    Furuta YHunter NBarkley T JrHall EMilas L: Increase in radioresponse of murine tumors by treatment with indomethacin. Cancer Res 48:300830131988

  • 14

    Galli RBinda EOrfanelli UCipelletti BGritti ADe Vitis S: Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64:701170212004

  • 15

    Gasparini GLongo RSarmiento RMorabito A: Inhibitors of cyclo-oxygenase 2: a new class of anticancer agents?. Lancet Oncol 4:6056152003

  • 16

    Grösch SMaier TJSchiffmann SGeisslinger G: Cyclooxy-genase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst 98:7367472006

  • 17

    Hide TTakezaki TNakamura HKuratsu JKondo T: Brain tumor stem cells as research and treatment targets. Brain Tumor Pathol 25:67722008

  • 18

    Kang KBWang TTWoon CTCheah ESMoore XLZhu C: Enhancement of glioblastoma radioresponse by a selective COX-2 inhibitor celecoxib: inhibition of tumor angiogenesis with extensive tumor necrosis. Int J Radiat Oncol Biol Phys 67:8888962007

  • 19

    Kao CLHuang PITsai PHTsai MLLo JFLee YY: Resveratrol-induced apoptosis and increased radiosensitivity in CD133-positive cells derived from atypical teratoid/rhabdoid tumor. Int J Radiat Oncol Biol Phys 74:2192282009

  • 20

    Kawabe T: G2 checkpoint abrogators as anticancer drugs. Mol Cancer Ther 3:5135192004

  • 21

    Kesari SSchiff DHenson JWMuzikansky AGigas DCDoherty L: Phase II study of temozolomide, thalidomide, and celecoxib for newly diagnosed glioblastoma in adults. Neuro Oncol 10:3003082008

  • 22

    Knizetova PDarling JLBartek J: Vascular endothelial growth factor in astroglioma stem cell biology and response to therapy. J Cell Mol Med 12:1111252008

  • 23

    Kuipers GKSlotman BJWedekind LEStoter TRBerg JSminia P: Radiosensitization of human glioma cells by cyclooxygenase-2 (COX-2) inhibition: independent on COX-2 expression and dependent on the COX-2 inhibitor and sequence of administration. Int J Radiat Biol 83:6776852007

  • 24

    Liao ZKomaki RMilas LYuan CKies MChang JY: A phase I clinical trial of thoracic radiotherapy and concurrent celecoxib for patients with unfavorable performance status inoperable/unresectable non-small cell lung cancer. Clin Cancer Res 11:334233482005

  • 25

    Lin CPTseng WYCheng HCChen JH: Validation of diffusion tensor magnetic resonance axonal fiber imaging with registered manganese-enhanced optic tracts. Neuroimage 14:103510472001

  • 26

    Liu WChen YWang WKeng PFinkelstein JHu D: Combination of radiation and celebrex (celecoxib) reduce mammary and lung tumor growth. Am J Clin Oncol 26:S103S1092003

  • 27

    Ma SChan KWHu LLee TKWo JYNg IO: Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 132:254225562007

  • 28

    Mantovani GMacciò AMadeddu CSerpe RAntoni GMassa E: Phase II nonrandomized study of the efficacy and safety of COX-2 inhibitor celecoxib on patients with cancer cachexia. J Mol Med 88:85922010

  • 29

    McAdam BFCatella-Lawson FMardini IAKapoor SLawson JAFitzGerald GA: Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2. Proc Natl Acad Sci U S A 96:2722771999

  • 30

    Miki JFurusato BLi HGu YTakahashi HEgawa S: Identification of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized primary nonmalignant and malignant tumor-derived human prostate epithelial cell lines and in prostate cancer specimens. Cancer Res 67:315331612007

  • 31

    Mizrak DBrittan MAlison MR: CD133: molecule of the moment. J Pathol 214:392008

  • 32

    Mutter RLu BCarbone DPCsiki IMoretti LJohnson DH: A phase II study of celecoxib in combination with paclitaxel, carboplatin, and radiotherapy for patients with inoperable stage IIIA/B non-small cell lung cancer. Clin Cancer Res 15:215821652009

  • 33

    Nakata EMason KAHunter NHusain ARaju ULiao Z: Potentiation of tumor response to radiation or chemoradiation by selective cyclooxygenase-2 enzyme inhibitors. Int J Radiat Oncol Biol Phys 58:3693752004

  • 34

    Nurse P: Universal control mechanism regulating onset of M-phase. Nature 344:5035081990

  • 35

    Pawlik TMKeyomarsi K: Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys 59:9289422004

  • 36

    Ricci-Vitiani LLombardi DGPilozzi EBiffoni MTodaro MPeschle C: Identification and expansion of human colon-cancer-initiating cells. Nature 445:1111152007

  • 37

    Sheng HShao JKirkland SCIsakson PCoffey RJMorrow J: Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2. J Clin Invest 99:225422591997

  • 38

    Singh SKHawkins CClarke IDSquire JABayani JHide T: Identification of human brain tumour initiating cells. Nature 432:3964012004

  • 39

    Smits VAMedema RH: Checking out the G(2)/M transition. Biochim Biophys Acta 1519:1122001

  • 40

    Stupp RHegi MEGilbert MRChakravarti A: Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 25:412741362007

  • 41

    Stupp RMason WPvan den Bent MJWeller MFisher BTaphoorn MJ: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:9879962005

  • 42

    Tang CChua CLAng BT: Insights into the cancer stem cell model of glioma tumorigenesis. Ann Acad Med Singapore 36:3523572007

  • 43

    Zeppernick FAhmadi RCampos BDictus CHelmke BMBecker N: Stem cell marker CD133 affects clinical outcome in glioma patients. Clin Cancer Res 14:1231292008

  • 44

    Zhu LGibson PCurrle DSTong YRichardson RJBayazitov IT: Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature 457:6036072009

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