Biology contribution
Enhancement of glioblastoma radioresponse by a selective COX-2 inhibitor celecoxib: Inhibition of tumor angiogenesis with extensive tumor necrosis

Presented at 97th Annual Meeting of the American Association for Cancer Research (AACR), April 1–5, 2006, Washington, D.C.
https://doi.org/10.1016/j.ijrobp.2006.09.055Get rights and content

Purpose: Toward improved glioblastoma multiforme treatment, we determined whether celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, could enhance glioblastoma radiosensitivity by inducing tumor necrosis and inhibiting tumor angiogenesis.

Methods and Materials: U-87MG cells treated with celecoxib, irradiation, or both were assayed for clonogenic survival and angiogenic factor protein analysis (angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor [VEGF]). In vivo, survival of mice intracranially implanted with U-87MG cells and treated with celecoxib and/or irradiation was monitored. Isolated tumors were assessed for tumor necrosis and tumor microvascular density by von Williebrand’s factor (vWF) immunohistochemical staining.

Results: Celecoxib (4 and 30 μM; 24, 48, and 72 h) enhanced U-87MG cell radiosensitivity by significantly reducing clonogenic survival of irradiated cells. Angiopoietin-1 and VEGF proteins were decreased, whereas angiopoietin-2 expression increased after 72 h of celecoxib alone and when combined with irradiation. In vivo, median survival of control mice intracranially implanted with U-87MG cells was 18 days. Celecoxib (100 mg/kg/day, 2 weeks) significantly extended median survival of irradiated mice (24 Gy total) from 34 to 41 days, with extensive tumor necrosis [24.5 ± 8.6% of tumor region, compared with irradiation alone (2.7 ± 1.8%)]. Tumor microvascular density was significantly reduced in combined celecoxib and irradiated tumors (52.5 ± 2.9 microvessels per mm2 tumor region), compared with irradiated tumors alone (65.4 ± 4.0 microvessels per mm2).

Conclusion: Celecoxib significantly enhanced glioblastoma radiosensitivity, reduced clonogenic survival, and prolonged survival of glioblastoma-implanted mice by inhibition of tumor angiogenesis with extensive tumor necrosis.

Introduction

Due to the complex molecular pathogenesis of glioblastoma multiforme, therapeutic strategies are evolving from cytotoxic therapies to molecular approaches which target specific signaling cascades that promote tumor growth such as cyclooxygenase-2 (COX-2), a rate-limiting enzyme in conversion of arachidonic acid into prostaglandins (1). Two COX isoforms (COX-1 and COX-2) have been identified, and COX-3, a splice variant of COX-1, has recently been proposed (2). The constitutive COX-1 isoform produces prostaglandins responsible for maintenance of gastrointestinal mucosa, vascular tone, and kidney and platelet function. COX-2 is induced by inflammatory stimuli including cytokines and growth factors and is associated with inflammatory diseases and carcinogenesis (3). High COX-2 overexpression has been detected in various tumors, with regression of tumor growth in the presence of COX-2 inhibitors (4, 5). In brain tumors, greater COX-2 expression is associated with high-grade glioblastomas (compared with low-grade tumors) and is a strong predictor of poor survival (6, 7). Selective COX-2 inhibition by celecoxib, SC-236, and NS-398 reduced human glioblastoma cell viability in vitro (6, 8) and when transplanted into rodents in vivo (9, 10, 11, 12).

The combined effect of a selective COX-2 inhibitor and radiotherapy holds much promise for antitumor benefit. COX-2 is overexpressed in irradiated tumor cells (13) and is a molecular marker of radioresistance (14). Increased level of prostaglandins after irradiation is radioprotective, promoting tumor growth by stimulation of cell proliferation, reduction of cell apoptosis (15), and enhanced DNA repair mechanisms (16). Enhanced tumor radiosensitivity by COX-2 inhibitors has become evident in various tumors. In vitro, NS-398 and SC-236 enhanced radiation-induced tumor cell death in NMF11.2 mouse mammary tumors (17), HEp3 head and neck squamous cell carcinomas (18), H460 human lung cancer cells (19), and NFSA murine fibrosarcoma cells (20). In vivo, mice hind legs transplanted with lung carcinoma (21), colon carcinoma (22), and sarcoma (23) cells, showed enhanced suppression of tumor growth rate by combination of radiotherapy with COX-2 inhibitors compared with single therapies. However, studies to investigate the combined effect of COX-2 inhibition and radiotherapy in brain tumor models are limited. The only published report by Petersen et al. (9) showed enhanced tumor radiosensitivity by SC-236 in U251 human glioblastoma cells, grown as monolayer culture and as tumor xenograft in mice thighs. Here we report the effect of celecoxib on tumor radioresponse of nude mice intracranially implanted with U-87MG glioblastoma cells.

The underlying mechanisms for enhanced tumor radioresponse by COX-2 inhibitors have not been defined. Proposed mechanisms include increased susceptibility to apoptosis induction (9, 19), enhanced G2M phase cell cycle arrest (20), inhibition of repair from sublethal radiation damage (9), inhibition of prostaglandin-induced immunosuppressive activity (24), and inhibition of angiogenesis (23). Angiogenesis, which critically supports the growth of glioblastoma multiforme, is a hallmark of these tumors. To elucidate whether celecoxib enhanced glioblastoma radiosensitivity by inhibiting tumor angiogenesis, we measured prostaglandin E2, vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2 levels, as well as tumor necrosis and microvascular density, following combined celecoxib and radiotherapy treatment. Our results demonstrated that celecoxib enhanced glioblastoma radiosensitivity of brain tumor mice, by inhibition of tumor angiogenesis with extensive tumor necrosis.

Section snippets

Cell culture

The human glioblastoma cell line U-87MG was obtained from American Type Culture Collection (Rockville, MD). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS, 10%), nonessential amino acid (100 μM), sodium pyruvate (1 mM), streptomycin (100 μg/mL), and penicillin (100 U/mL, Gibco BRL, Grand Island, NY) at 37°C in an atmosphere containing 5% CO2.

Drug treatment and irradiation

Cells were starved (in 1% FBS media) overnight before treatment with celecoxib (Pfizer Pharmacia,

Celecoxib reduced U-87MG cell viability

Celecoxib dose-dependently reduced the viability of U-87MG cells with EC50 values of 39.3, 34.7, and 33.5 μM, at 24, 48, and 72 h, respectively (data not shown). The U.S. Food and Drug Administration (FDA)-approved celecoxib dosage for patients with familial adenomatous polyposis is 800 mg/day, corresponding to a peak plasma concentration of 4 μM (30). From the clinically relevant concentration of celecoxib and the EC50 celecoxib concentration data, we derived our study based on celecoxib

Discussion

Our study showed that celecoxib enhanced U87-MG glioblastoma radiosensitivity, as demonstrated by the significant reduction in clonogenic survival in vitro and extended median survival of glioblastoma-implanted mice. To the best of our knowledge, this is the first report to demonstrate enhancement of tumor radiosensitivity by celecoxib at clinically relevant concentrations in mice with glioblastoma-implanted brain tumor. In a different glioblastoma model, Petersen et al., (2000) showed that the

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