Annals of Oncology Advance Access originally published online on September 28, 2006
Annals of Oncology 2006 17(11):1625-1630; doi:10.1093/annonc/mdl283
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© 2006 European Society for Medical Oncology
head and neck cancer |
Celecoxib reduces microvessel density in patients treated with nasopharyngeal carcinoma and induces changes in gene expression
1 Department of Haematology–Oncology, National University Hospital, Singapore
2 Department of Medicine, National University Hospital, Singapore
3 Department of Otolaryngology–Head and Neck Surgery, National University Hospital, Singapore
4 Cellular and Molecular Research, National Cancer Centre, Singapore
5 Cancer Epigenetics/Tumor Virology Laboratory, Prince of Wales Hospital, Hong Kong
6 Tumor Virology Laboratory, Johns Hopkins in Singapore, Singapore
7 Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
* Correspondence to: Dr R. A. Soo, Department of Haematology–Oncology, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074. Tel: +65 6772 4621; Fax: +65 6777 5545; E-mail: Ross_SOO{at}nuh.com.sg
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Abstract |
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Background: Celecoxib is a selective cyclooxygenase-2 inhibitor with antitumor and antiangiogenic activity. We sought to determine pharmacodynamic change in tumors of patients with nasopharyngeal carcinoma (NPC) treated with celecoxib.
Methods: Tumor biopsies were obtained before and after treatment with celecoxib 400 mg b.i.d. for 14 days in patients with newly diagnosed, untreated NPC. Tumor angiogenesis and cell proliferation were assessed by immunohistochemistry and gene expression by microarray analysis. Plasma celecoxib concentrations were obtained on days 8 and 14.
Results: Paired samples were analyzed in 15 patients. Microvessel density was reduced in post-treatment samples and mean celecoxib levels reached therapeutic levels. Thirty-five genes (27 down-regulated, eight up-regulated) were differentially expressed on microarray analysis (p < 0.001). Down-regulated genes included cell cycle regulation-related (cyclin-dependent kinase 2, YES1), transcription factor (TRIP-Br2), whereas the antigen processing and presentation-related gene HLA-DM B was up-regulated.
Conclusion: Celecoxib reduced angiogenesis and induced tumor transcriptional changes. Further characterization of these transcriptional changes in vivo is needed to provide further insights into the effects of celecoxib in neoplastic tissue. Our findings provide a rationale for clinical studies aimed at assessing the efficacy of celecoxib in the treatment of NPC.
Key words: antiangiogenesis, celecoxib, gene expression, nasopharyngeal carcinoma
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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Cyclooxygenase (COX), a key enzyme in prostaglandin synthesis, exists in two isoforms: COX-1 and COX-2. COX-1, a housekeeping enzyme is expressed constitutively and is responsible for the production of prostaglandins that mediate normal physiological function [1]. COX-2, undetectable in most normal tissues, is induced by cytokines, tumor promoters and growth factors and appears to have an important role in inflammation and carcinogenesis [2]. COX-2 is overexpressed in malignancy and is associated with poorer prognosis [3]. In preclinical studies, COX-2 is associated with tumorigenesis, angiogenesis and inhibition of apoptosis [3]. COX-2 deletion in mice susceptible to adenomatous polyposis markedly reduced intestinal polyposis [4], while COX-2 overexpression increased mammary tumors [5].
World Health Organization type III or undifferentiated-type nasopharyngeal carcinoma (NPC) is endemic to many areas of Asia [6] and is the fifth most common cause of cancer deaths among males in Singapore [7]. Despite improved results with concurrent chemoradiation, significant numbers of patients with locally advanced disease still relapse after treatment and patients with metastatic disease are not cured with conventional treatment. COX-2 is overexpressed in 71%–79% of NPC by immunohistochemistry (IHC) and angiogenesis is increased [8, 9]. The presence of angiogenesis is associated with local invasion, nodal and systemic metastases [10, 11] and poorer prognosis [12, 13].
In preclinical studies, celecoxib (Celebrex®; Pfizer, New York), a COX-2 inhibitor, has the ability to induce apoptosis and reduce cell proliferation and angiogenesis [14–16]. Studies using NPC cell lines have found that celecoxib induced cell apoptosis and cell cycle arrest through down-regulation of cyclin D expression and suppressed genes related to angiogenesis [17–19]. In a randomized study of patients with familial adenomatous polyposis administered celecoxib 400 mg b.i.d. for 6 months, the number of colorectal polyps was significantly reduced [20].
We conducted a trial using a window study design in patients with NPC to elucidate the changes in angiogenesis in response to brief celecoxib exposure. Given the potential for interruption of multiple tumorigenic pathways through COX-2 inhibition and the possibility of interaction between tumor and supporting stromal cells, we also sought to profile gene expression changes following celecoxib exposure.
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patients and methods |
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patients
Patients aged
18 years with histologically confirmed, untreated NPC were eligible. Patients with a known contraindication to celecoxib, unable to take celecoxib for the 14 days before definitive anticancer therapy or if the tumor was not visible on nasopharyngoscopy for biopsy; with a history of a second primary cancer; or who were pregnant or lactating or had received nonsteroidal anti-inflammatory drugs or corticosteroids within 4 weeks before study entry were excluded from the study. Patients received celecoxib 400 mg b.i.d. for 14 days. NPC biopsies were obtained before treatment and on day 14 of celecoxib. Paired tumor biopsies from five patients not treated with celecoxib were used as controls as the biopsy process may modify markers of interest. All samples were immediately placed in liquid nitrogen after biopsy and stored at –80°C until required. Written informed consent was obtained from all patients and the institutional review board approved the study.
immunohistochemistry
Cell proliferation and tumor angiogenesis were measured by Ki-67 and microvessel density (MVD) [with anti-CD31 monoclonal antibody (mAb)], respectively. COX-2 expression was also measured. Hematoxylin–eosin staining for one tissue section was done for histological review and the remaining sections were immunostained with anti-COX-2 (Cayman Chemicals, Ann Arbor, MI), anti-CD-31 (Dako A/S, Glostrup, Denmark) and Ki-67 mAbs (MIB-1 clone) (Dako A/S). Microwave antigen retrieval was employed and further processing involved the use of the EnVision Kit (Dako A/S). Omission of the primary antibodies and their replacement with normal serum was used as a negative control. Previously studied cases of breast carcinoma and lymph node tissue were used as positive controls for COX-2 and Ki-67, respectively. COX-2 expression and MVD were semiquantitatively assessed by the extent and intensity of staining as described previously [8, 9]. In brief, the extent score was assessed on a scale of 0–3, where 0 = negative, 1 = positive staining in <10% of cells, 2 = positive staining in 10%–50% of cells and 3 = positive staining in >50%. The intensity score was measured on a scale of 0–3, where 0 = negative, 1 = weak, 2 = moderate and 3 = strong. The immunoreactive score was obtained by multiplying the extent score with intensity score and a score of 4 was considered to be overexpressing COX-2. Tumor MVD was counted per high-power field at x400 magnification (x40 objective and x10 ocular). The tumor was scanned at lower power and the areas with the highest MVD were assessed in detail, excluding blood vessels located at the tumor edges. Microvessel counts of the two high-power fields with the highest MVD were obtained and the average per high-power field was determined. For Ki-67 evaluation, all fields of the tumor section were examined and the percentage of tumor nuclei with positive staining for Ki-67 was estimated. Scoring of each parameter was carried out in a blinded manner. Disagreement was noted in one case for Ki-67 and two for MVD. Consensus was reached by joint re-evaluation.
microarray hybridization
Total RNA was extracted from tumor tissue by Tri reagent (Molecular Research Center, Inc., Cincinnati, OH), purified through a Qiagen Spin Column (Chatsworth, CA) and processed for Affymetrix GeneChip (Affymetrix, Santa Clara, CA) hybridization according to the manufacturer's instructions. RNA amplification was not performed. For each sample, a starting amount of 10 µg total RNA was used and hybridized to oligonucleotide arrays (HG-U133A GeneChip, Affymetrix, Santa Clara, CA) that contained 22 283 transcripts and control sequences. The arrays were stained using the GeneChip Hybridization, Wash and Stain Kit (Affymetrix, Santa Clara, CA). Fluorescence intensities were subsequently determined using GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA). The signal intensity for each gene was obtained by applying to each GeneChip the MAS5 condensing algorithm, found in the Microarray Suite software package (Affymetrix, Santa Clara, CA).
gene expression data processing and statistical analysis
The raw GeneChip scans were quality controlled using Genedata Refiner software to identify and correct areas of uneven hybridization. The refined scans were deposited into a central data storage facility and data analysis was carried out using Genedata Expressionist software and conventional spreadsheet applications. The expression data were preprocessed by normalizing the expression profiles by median centering each array profile at 600 fluorescence units, and then removing the genes whose expression was absent in >60% of samples. This resulted in a starting expression dataset of 
10 341 genes. Average linkage hierarchical clustering employing Pearson correlation metrics was carried out on a truncated dataset consisting of 1742 genes exhibiting a minimal variance of 0.2 across all 10 341 genes. Minor alterations of this variation filter (0.1–0.4) gave highly similar results, as did principal component analysis of this gene set. Gene expression fold changes were presented as a ratio of post/pre-celecoxib therapy expression. To identify significantly differentially expressed genes in each paired patient sample, expression data on the post-celecoxib array were compared with the pre-celecoxib array using paired Wilcoxon test. A P <0.001 was considered statistically significant.
Paired Wilcoxon test was used to analyze the differences in Ki-67, and MVD in pre- and post-treatment biopsies and in controls. Results were considered significant at P < 0.05.
semiquantitative RT-PCR
RT-PCR for major histocompatibility complex, class II, DM beta (HLA-DMB), v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES1), cyclin-dependent kinase 2 (CDK2), tumor necrosis factor receptor superfamily, member 10b (TNFRSF10B), Transcriptional Regulator Interacting with the PHD zinc finger and/or the Bromodomain (TRIP Br2), NK3 transcription factor related, locus 1 (NKX3-1), and Transferrin Receptor 1 (TFRC) genes was carreid out using AmpliTaq Gold (Applied Biosystems, Foster City, CA) as described previously [21] using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a control, and its primers were designed according to the GAPDH mRNA sequence (National Center for Biotechnology Information nucleotide database accession No.: M33197). The PCR program started with an initial denaturation at 95°C for 10 min, followed by multiple cycles (94°C for 30 s, 51°C or 60°C for 30 s and 72°C for 30 s) of amplification, with a final extension at 72°C for 10 min. PCR bands were visualized under UV light, photographed and semiquantified with the EagleEye digital gel documentation system and the supplied densitometry software (EagleSight Software, version 3.2, Stratagene, La Jolla, CA). Twenty-five to thirty cycles of PCR were used for semiquantitative RT-PCR analysis. Sequences of gene-specific primers are available upon request.
celecoxib concentration levels
Blood samples (10 ml) were obtained at baseline (day 0), pre-dose and 2 h post-dose on days 8 and 14. Plasma celecoxib concentrations were determined by high-performance liquid chromatography with mass spectrometric detection at a commercial facility (PPD, Richmond, VA).
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results |
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patients
Thirty-four patients were recruited between March 2002 and July 2003. Eight did not complete the study: one withdrew consent, three were noncompliant and four developed a skin rash and were withdrawn from the study. No other adverse events were noted. In particular, as celecoxib use is associated with increased risk of cardiovascular events [22], no cardiovascular toxic effects were reported. One patient refused a second biopsy on day 15; thus, pre- and post-treatment paired biopsies were carried out in 25 patients. Of the remaining 15 paired samples, histologically confirmed NPC was present in at least 50% of each biopsy sample and was further analyzed.
The median age was 47 years (range 26–72 years) and 12 patients were male. Three patients had stage I or II disease, seven had stage III and five had stage IV (nonmetastatic) disease according to the 1997 American Joint Committee on Cancer staging and all had undifferentiated-type NPC (Tables 1 and 2).
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immunohistochemical analysis of samples
COX-2 was overexpressed in 13 of 15 (87%) of the pre-treatment samples and was similar following celecoxib therapy (Table 1). COX-2 was found within the tumor cell cytoplasm, whereas normal nasopharyngeal epithelium, vascular endothelium and stromal cells did not stain for COX-2 (Figure 1). The median MVD scores in the pre-treatment samples was 24 (range 17–34) and post-treatment was 17 (range 12–27, P = 0.001). The median percentage of Ki-67-positive cells before treatment was 25 (range 1–80) and after treatment was 20 (range 5–75) (P = 0.39) (Figure 2A and B). In patients not treated with celecoxib, COX-2 expression in tumor biopsies at baseline and 14 days later revealed no change (Table 2). MVD and Ki-67 expression were unaltered with MVD scores of 15 (range 5–20) and 16 (range 10–35) at baseline and after 14 days, respectively (P = 0.35); the median percentage of Ki-67-positive cells at baseline was 50 (range 5–50) and after 14 days was 20 (range 10–50) (P = 0.47).
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transcriptional changes with celecoxib treatment
RNA was extracted from the 15 paired samples to generate gene expression profiles. An unsupervised average linkage hierarchical clustering algorithm was used first to group the expression profiles to one another on the basis of their overall similarity. In eight of 15 cases, the pre- and post-treatment profiles derived from the same patient displayed similarities to one another than to any of the other samples (data not shown). To further characterize the gene expression changes in NPC following celecoxib treatment, Wilcoxon test was utilized and 35 transcripts (27 down-regulated, eight up-regulated) were significantly differentially expressed (P < 0.001) between the pre- and post-treatment (Table 3). Genes down-regulated were involved in cell growth/maintenance (CDK2, YES1), cell metabolism, cell signaling (frizzled homolog 6) and transcriptional regulation (NKX3-1, TRIP Br2), whilst up-regulated genes included genes involved in immune response (HLA-DMB). The median fold change of the post/pre expression ratio of these genes ranged from 0.7 to 1.9. Verification of the microarray results was performed using semi-quantitative RT-PCR in seven selected genes. These genes were selected based on their putative role in oncogenesis whilst the patient samples were based on the availability of adequate RNA in the samples. Consistent with the microarray data, the levels of YES1, TNFRSF10, CDK2, TRIP-Br2, and TFRC were reduced whilst HLA-DM II and NKX3-1 were increased post-treatment (Figure 3).
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celecoxib concentration levels
Plasma celecoxib concentrations on days 8 and 14 were analyzed in 30 and 25 patients, respectively. Pre-dose and 2-h post-dose celecoxib plasma levels on day 8 were 656 ± 372 ng/ml and 1797 ± 807 ng/ml and on day 14 were 669 ± 426 ng/ml and 1930 ± 889 ng/ml, respectively.
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discussion |
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Angiogenesis is associated with increased risk of local invasion, metastases and poorer prognosis in NPC. Celecoxib is known to induce pleiotrophic effects in tumor, and this work provides the first evidence of a reduction in angiogenesis as measured by MVD in patients with previously untreated NPC. In addition, multiple perturbations in gene expression with relatively short exposure to celecoxib were observed. Some of these differentially expressed genes are involved in cell signaling, metabolism, immunoregulation and transcription.
The antiangiogenic effect of short-term treatment with celecoxib in our study confirms a previous study reported in patients with cervical cancer in which a decrease in MVD was observed after treatment with celecoxib 400 mg b.i.d. for 10 days [23]. The reduction in MVD is unlikely to be related to the biopsy process, as MVD was unchanged in control samples. These observations are hypothesis generating in nature and serve as the basis for further investigations into the relationship between celecoxib and the reported differentially expressed genes. CDK2, YES1, and TRIP-Br2 were down-regulated. CDK2, a key cell cycle regulator, is over-expressed in some tumors [28] and YES1, a member of src family of tyrosine kinase, is also associated with a range of tumors [29] and chemoresistance [30]. TRIP-Br2, also known as SERTAD, is a member of the TRIP-Br family of transcription factors. The TRIP-Br proteins, TRIP-Br1 and -Br2, interact functionally with DP-1, resulting in the stimulation of E2F-1/DP-1 transcriptional activity and may have a role in E2F-dependent cell cycle progression [31]. NKX3-1 and HLA-DMB were up-regulated. NKX3-1 encodes for a transcription factor and is a putative prostate tumor suppressor gene [32]. HLA-DMB promotes peptide loading of class II molecules [33] and low levels of HLA-DM expression on APC may impair the development of adequate anti-tumor T cell response.
In this study, the gene TNFRSF10B that encodes for a known pro-apoptotic factor, tumor necrosis factor receptor superfamily, member 10b, was down-regulated.
As NPC tumors are relatively small, our study was limited by the small amount of tumor that could be safely obtained per individual, precluding further assays to determine intratumoral prostaglandin E2 levels and limiting the number of genes on which validation studies with semiquantitative RT-PCR could be carried out. With the available samples, we were able to confirm the gene expression changes with semiquantitative RT-PCR.
As angiogenesis has an important role in the biology of NPC, the finding that celecoxib reduced angiogenesis provides a rationale for clinical studies aimed at assessing the efficacy of celecoxib in the treatment of NPC. Differentially expressed genes identified in this study suggest further avenues of investigation which may further elucidate the mechanisms by which celecoxib exerts antitumor effects. Further studies characterizing gene expression changes by celecoxib in patients with malignant tumors are warranted.
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Supported by National Healthcare Group Grant NHG-RPR/01081 (RS) and Biomedical Research Council Grants BMRC 01/01/26/18/060 (B-CG), BMRC 01/01/31/19/209 (PT) and Agency for Science, Technology and Research (QT and WH).
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Footnotes |
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These authors contributed equally to this work. 
Received for publication January 16, 2006. Revision received June 28, 2006. Accepted for publication July 3, 2006.
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