Biology Contribution
Celecoxib Induced Tumor Cell Radiosensitization by Inhibiting Radiation Induced Nuclear EGFR Transport and DNA-Repair: A COX-2 Independent Mechanism

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Purpose

The purpose of the study was to elucidate the molecular mechanisms mediating radiosensitization of human tumor cells by the selective cyclooxygenase (COX)-2 inhibitor celecoxib.

Methods and Materials

Experiments were performed using bronchial carcinoma cells A549, transformed fibroblasts HH4dd, the FaDu head-and-neck tumor cells, the colon carcinoma cells HCT116, and normal fibroblasts HSF7. Effects of celecoxib treatment were assessed by clonogenic cell survival, Western analysis, and quantification of residual DNA damage by γH2AX foci assay.

Results

Celecoxib treatment resulted in a pronounced radiosensitization of A549, HCT116, and HSF7 cells, whereas FaDu and HH4dd cells were not radiosensitized. The observed radiosensitization could neither be correlated with basal COX-2 expression pattern nor with basal production of prostaglandin E2, but was depended on the ability of celecoxib to inhibit basal and radiation-induced nuclear transport of epidermal growth factor receptor (EGFR). The nuclear EGFR transport was strongly inhibited in A549-, HSF7-, and COX-2–deficient HCT116 cells, which were radiosensitized, but not in FaDu and HH4dd cells, which resisted celecoxib-induced radiosensitization. Celecoxib inhibited radiation-induced DNA-PK activation in A549, HSF7, and HCT116 cells, but not in FaDu and HH4dd cells. Consequentially, celecoxib increased residual γH2AX foci after irradiation, demonstrating that inhibition of DNA repair has occurred in responsive A549, HCT116, and HSF7 cells only.

Conclusions

Celecoxib enhanced radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance, a signaling that was independent of COX-2 activity. This novel observation may have therapeutic implications such that COX-2 inhibitors may improve therapeutic efficacy of radiation even in patients whose tumor radioresistance is not dependent on COX-2.

Introduction

Celecoxib belongs to the group of specific cyclooxygenase 2 (COX-2) inhibitors that originally were approved by the Food and Drug Administration for treatment of rheumatoid arthritis. COX-2 inhibitors were found to be effective also in prevention of development of intestinal 1, 2 and skin cancers (3). Therapeutic benefit can be achieved by combining celecoxib with radiation and chemotherapy during treatment of glioblastomas (4), prostate carcinomas (5), and lung cancer 6, 7. Taken together, there exists solid evidence that inhibition of COX-2 with selective inhibitors prevents carcinogenesis, slows the growth of established tumors, and enhances tumor response to radiation without appreciably affecting normal tissue radioresponse 8, 9. Albeit intensive investigations to understand the cellular consequences of celecoxib treatment, it has not been fully resolved which molecular processes are involved in radiosensitization. Celecoxib and other COX-2 selective inhibitors inhibit proliferation of and induce apoptosis in a variety of tumor cells 10, 11. They affect some normal cells as well. For example, at high concentrations, both rofecoxib and celecoxib induce apoptosis in endothelial cells, but do not effect either the induction of apoptosis or inhibition of cell-cycle progression in human vascular smooth muscle cells 11, 12, suggesting a cell type–dependency of these effects of COX-2 inhibitors. Some other effects of COX-2 inhibitors such as reduction of angiogenesis and inhibition of invasiveness of tumor cells may also be cell type–dependent (13).

At the molecular level, COX-2 inhibitors affect signal transduction pathways involved in cell proliferation, cell-cycle, and apoptosis development 14, 15. Among these is the phosphatidylinositol 3-kinase/Akt pathway reported recently to be affected by celecoxib 16, 17, 18. The exact signaling for phosphatidylinositol 3-kinase/Akt activation is unclear, but some recent findings suggest that both PGE2, a major metabolite of COX-2, and epidermal growth factor receptor (EGFR) are involved (17). Buchanan et al.(17) found that PGE2-induced migration and invasion involved rapid transactivation and phosphorylation of EGFR. Within minutes after treatment, PGE2 induced activation of Akt, which was completely abolished by EGFR-specific tyrosine kinase inhibitors, providing evidence for a distinct role of EGFR during this process. It was reasoned that the rapid transactivation of EGFR occurred probably via an intracellular Src-mediated event without release of extracellular epidermal growth factor-like ligand (17). Ionizing radiation is also able to induce EGFR transactivation (18), in which case this molecular process was found to be essential for cell survival 19, 20. The crucial role of EGFR during cell survival after exposure to radiation or some other cellular stresses is accentuated by our recent observation (21). We found that EGFR became translocated into the cell nucleus and regulated DNA-PK activity essential for repair of DNA double-strand breaks (21).

The antitumor effects of COX-2 inhibitors, used either as single treatments or in combination with radiation or chemotherapeutic agents, have commonly been attributed to selective COX-2 inhibitory activities of coxibs 8, 9. However, increasing evidence suggests that these agents may exert antitumor actions by COX-2 independent mechanisms as well. Reports show that these agents (1) inhibit in vitro growth of cells (22) and in vivo growth of tumors (23), lacking COX-2; (2) need to be given in higher doses to block cell proliferation than those that inhibit the enzyme (24); and (3) retain inhibitory properties for cells in vitro and tumors in vivo when derivatives of celecoxib lack COX-2 suppressive activity 4, 25.

Using cell lines expressing or lacking COX-2, the present study assessed the effect of celecoxib on radiosensitivity and molecular mechanisms underlying celecoxib-induced radiosensitization, concentrating on the involvement of EGFR. Data presented show that celecoxib inhibited radiation-induced nuclear translocation of EGFR in responding cells, which was associated with inhibition of DNA repair processes, and thus provide a novel explanation for the radiosensitizing effects of COX-2 inhibiting agents.

Section snippets

Cell culture and irradiation

Celecoxib was kindly provided by Pfizer Corp. (Karlsruhe, FRG). Human bronchial carcinoma cells, designated A549 (26), normal foreskin fibroblasts HSF7, transformed fibroblasts HH4dd (27), HCT116 cells (ATCC, origin colon carcinoma), and the human squamous carcinoma cell line FaDu (ATCC, origin head-and-neck cancer) were used. Cells were trypsinized and seeded for colony formation assay in 78 cm2 plates. Twenty-four hours after plating cells were treated with celecoxib (10 μM) for 48 hours and

Radiosensitization of tumor cells and normal cells by celecoxib

Celecoxib pretreatment in a clinical relevant dose of 10 μM led to a clear radiosensitization in the human bronchial adenocarcinoma cell line A549 (surviving fraction at 2 Gy (SF2) = 0.57 vs. 0.39 in presence of celecoxib, dose enhancement rate = 1.46) (Fig. 1a). Likewise, the normal fibroblast cell strain HSF7 (SF2 = 0.42 vs. 0.16) and the colon carcinoma cell line HCT116 (SF2 = 0.72 vs. 0.55) responded to celecoxib treatment with a clear radiosensitization (Fig. 1c, e). In contrast, the human

Discussion

The data presented herein clearly demonstrate a differential effect of combined celecoxib/radiation treatment on clonogenic survival of human tumor cells and normal fibroblasts in vitro. Unlike in the bronchial cancer cell line A549, the colon carcinoma cell line HCT116 and normal fibroblasts responded with a marked radiosensitization, the head-and-neck tumor cell line FaDu as well as the tumorigenic, transformed fibroblast line HH4dd did not respond. These differential effects seem to be

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  • Cited by (0)

    Supported by Deutsche Krebshilfe Grant 106401, the National Institutes of Health (NIH) Grant CA06294 and National Cancer Institute (NCI) Cancer Center Core Grant CA16672.

    Conflicts of interest: none.

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