- Email: [email protected]
RETRACTED: Overexpression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis
Oral Oncology (2005) 41, 903–908
http://intl.elsevierhealth.com/journals/oron/
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Overexpression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis
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Jyh-Ping Peng a, Hui-Chiu Chang b, Chung-Feng Hwang a, Wen-Chun Hung b,* a
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Department of Otolaryngology, Chang Gung University, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Kaohsiung, Taiwan, ROC b Institute of Biomedical Sciences, National Sun Yat-Sen University, No. 70, Lien-Hai Road, Kaohsiung 804, Taiwan, ROC Received 20 April 2005; accepted 4 May 2005
KEYWORDS
Summary Cyclooxygenase-2 (COX-2) has been shown to be involved in multiple steps of carcinogenesis. In this study, we examined COX-2 expression in nasopharyngeal carcinoma (NPC). COX-2 mRNA analyzed by reverse-transcription polymerase chain reaction (RT-PCR) was detected in 66% (16 of 24) of tumor tissues. Western blot analysis demonstrated that COX-2 protein level was increased in tumor tissues and was correlated with the expression level of mRNA. Immunohistochemical study showed that COX-2 was predominantly detected in cancer cells, and the staining pattern was cytoplasmic. Ten histologically normal nasopharyngeal tissues obtained from nasopharyngeal hyperplasia were also investigated. We found that COX-2 mRNA was detectable in three tissues and the COX-2 protein level was very low. The frequency of COX-2 overexpression was significantly higher in patients of the N1–N3 group than in patients of the N0 group (P = 0.006). Taken together, these data suggest that COX-2 is overexpressed and is associated with increased lymphatic invasion in NPC. c 2005 Elsevier Ltd. All rights reserved.
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Cyclooxygenase-2; Nasopharyngeal carcinoma; Metastasis; RT-PCT; Western blot analysis; Immunohistochemical staining
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Introduction
* Corresponding author. Tel.: +886 7 5252000; fax: +886 7 5250197. E-mail address: [email protected] (W.-C. Hung).
Nasopharyngeal carcinoma (NPC) is characterized by its poor clinical outcome due to early metastasis and rapid progression. There is a high prevalence of NPC in Southeast Asia, southern China, and the Mediterranean basin.1 In endemic areas such as
1368-8375/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2005.05.003
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microgram of total RNA was reverse-transcribed to complementary DNA (cDNA) by use of the OneStep reverse-transcription polymerase chain reaction (RT-PCR) kit (Qiagen) according to the manufacturer’s protocol. Each PCR reaction was performed in 50 lL of a reaction mix containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.4 mM deoxynucleotide triphosphate, 600 nM primers, and 3 U of HotStarTaq DNA polymerase. Five microliters of the reverse-transcribed cDNAs was added to the reaction mix and amplified for 30 cycles of denaturation at 94 C for 45 s, annealing at 60 C for 45 s, extension at 72 C for 2 min, and final extension at 72 C for 10 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. The primer sequence and PCR product size for COX-2, 50 -GGTCTGGTGCCTGGTCTGATGATG-30 (sense) and 50 -GTCCTTTCAAGGAATGGTGC-30 (antisense), 724 base pairs (bp). PCR product was run on 2% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light.
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Taiwan, 90% of these squamous cell carcinomas are WHO type IIa or IIb, that are very sensitive to radiation.2,3 Previous epidemiologic studies have indicated that several etiological factors including exposure of chemical carcinogens (such like Cantonese salted fish), and Epstein–Barr virus (EBV) infection may play an important role in the development of NPC. However, the mechanism by which these factors contribute to the tumorigenesis of NPC is largely unknown. Prostaglandins (PGs) are involved in the regulation of various physiologic and pathophysiologic processes, including blood clotting, kidney function, wound healing, cardiovascular disease, and inflammation.4 Recent works show that the conversion of arachidonic acid to PGs is catalized by cyclooxygenases (COXs). Two isoforms of COX, COX-1 and COX-2, have been identified. COX-1 is expressed constitutively in various tissues. Conversely, COX-2 expression is undetectable in most tissues and is induced by mitogens, cytokines, or tumor promoters.5 Lines of evidence suggest that COX-2 is participated in the steps of carcinogenesis. First, increased expression of COX-2 was frequently found in human cancers, including gastric, esophageal, lung, and colon cancer.6–9 Second, Overexpression of COX-2 is sufficient to induce tumorigenesis in transgenic mice.10 Third, non-steroidal anti-inflammatory drugs (NSAIDs), potent inhibitors of COXs, have been shown to exert chemopreventive effect on cancer development.11,12 More importantly, upregulation of COX-2 in cancer cells has been shown to link with increased angiogenesis and metastasis.13,14 In this study, we examine COX-2 expression in NPC and try to address its correlation with tumor metastasis.
Materials and methods
Normal and NPC samples
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Tumor tissues were obtained from 24 patients who underwent resection of tumors at Chang Gung Memorial Hospital, Kaohsiung Medical Center. Tumor tissues were sharply excised, placed immediately in liquid nitrogen, and stored at 80 C until use. Ten histologically normal hyperplasia tissues were also collected by the same method.
RNA extraction and reverse-transcription polymerase chain reaction Total RNA was isolated from tissues using a RNeasy Mini Kits from Qiagen (Santa Clarita, CA). One
Western blot analysis
Tissues were homogenized in a tissue homogenization buffer (50 mM Tris–HCl, pH 7.4, 250 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 2 mM sodium orthovanadate, 50 mM NaF, 1 mM phenylmethylsulphonyl fluoride, 1 mg/ml aprotinin, 2 lg/ml pepstatin A, and 2 lg/ml leupeptin). Tissue homogenates were centrifuged at 12,000g for 20 min at 4 C, and protein concentrations were determined using a BCA protein assay kit (Pierce, IL). Equal amounts of cellular proteins were subjected to separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and Western blot analysis was performed as described previously.15 Tumor tissues were classified as overexpression of protein when the signal intensity of tumor tissues was two-fold higher than the average intensity of the ten histologically normal hyperplasia tissues.
Immunohistochemistry Immunohistochemical staining was performed as described previously.16 In brief, 5-lm frozen sections were fixed with acetone and treated with 0.5% hydrogen peroxide to remove endogenous peroxidase activity. Tissue sections were probed with COX-2 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C. Tissue sections were washed twice with phosphate-buffered saline (PBS) and incubated with
Overexpression of COX-2 in NPC and association with lymph node metastasis
The expression of COX-2 in relation to lymph node metastasis was analyzed by using the Fisher’s exact test. A difference between groups of P < 0.05 was considered significant.
Results
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COX-2 is overexpressed in NPC The patients had a mean age 51 (range 32–72) years and included 18 men and 6 women. The degree of differentiation of the tumors and patient’s T and N status are summarized in Table 1. Of the 24 NPC tissues analyzed by RT-PCR,
Expression of COX-2 in nasopharyngeal carcinoma Sex
Stage
Differentiationa (WHO type)
COX-2 mRNA
COX-2 protein level
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
51 33 66 41 71 41 43 41 46 53 63 48 51 45 72 53 66 60 62 66 56 32 32 33
M M M F M F F M M F M F M M M F M M M M M M M M
T1N2 T4N0 T2N1 T2N1 T4N1 T2N1 T3N0 T2N3 T3N1 T1N2 T2N1 T3N2 T1N2 T1N2 T3N0 T4N0 T2N1 T2N3 T1N2 T3N1 T1N2 T3N1 T2N1 T1N1
IIb IIa IIa IIa I IIa IIa IIa IIa IIa IIb IIb I IIa IIa IIa IIa IIa IIa IIa IIa IIa IIa IIb
+ + + + + + + + + + + + + + + +
Overexpression Undetectable Overexpression Overexpression Overexpression Overexpression Undetectable Overexpression Overexpression Overexpression Overexpression Overexpression Overexpression Overexpression Undetectable Undetectable Undetectable Overexpressed Overexpressed Undetectable Overexpressed Overexpressed Undetectable Undetectable
Normal 1 2 3 4 5 6 7 8 9 10
+ + +
Undetectable Undetectable Undetectable Undetectable Low expression Low expression Low expression Undetectable Undetectable Undetectable
a
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Age (years)
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Table 1
Statistical analysis
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biotinylated secondary antibody (Dako, Copenhagen, Denmark) for 30 min at room temperature. The sections were washed with PBS, incubated with peroxidase-labeled streptavidin, and then developed by 3-amino-9-ethylcarbazole. Finally, the specimens were counterstained with Mayer hematoxylin and mounted with Entellan (Merck, Darmstadt, Germany). Negative controls were carried out by replacing primary antibody with non-immune antiserum. The 24 NPC tissues were categorized as WHO type and the T-stage distribution was classified according to the AJCC classification. From World Health Organization histological study, NPC samples of distinct histological types, including 2 squamous cell carcinoma (WHO type I), 18 non-keratinizing carcinoma (WHO type IIa), and 4 undifferentiated carcinoma (WHO type IIb) were studied.
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nasopharyngeal 47 24 27 31 54 36 39 57 65 16
tissues M M M M M F M F F M
Type I, squamous cell carcinoma; type IIa, non-keratinizing carcinoma; type IIb, undifferentiated carcinoma.
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Figure 3 Immunohistochemical analysis shows positive cytoplasmic staining for COX-2 protein in NPC tissue (Immunohistochemistry · 100).
COX-2 overexpression is associated with lymph node metastasis
Pathological investigations have demonstrated that COX-2 expression is associated lymph node metastasis in breast, colon, cervical, gastric and endometric cancers.17–21 Therefore, we addressed the correlation between COX-2 expression and lymph node metastasis in NPC patients. Table 2 showed that the frequency of COX-2 expression was significantly higher in the N1–N3 group than in the N0 group (P = 0.006, Fisher exact test). Additionally, all of the tumor tissues of the N0 group were negative for COX-2 mRNA expression. These results suggest that overexpression of COX-2 is associated with increased lymphatic invasion in NPC.
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expression of COX-2 mRNA was detected in 16 (66%) samples (Fig. 1 and Table 1). We also investigated histologically normal nasopharyngeal hyperplasia tissues. Our results demonstrated that low level of COX-2 mRNA was detected in three normal tissues. We next investigated COX-2 protein level in normal and tumor tissues by Western blot analysis and the autoradiograms were subjected to densitometric analysis. Tumor tissues were classified as overexpression of COX-2 protein when the signal intensity of tumor tissues was two-fold higher than the average signal intensity of 10 normal tissues. Our data demonstrated that COX-2 was highly expressed and was correlated with mRNA level in NPC tissues (Fig. 2). All of the NPC tumor tissues positive for COX-2 mRNA are found to be overexpression of COX-2 protein in Western blot analysis. Conversely, the COX-2 protein level of the three normal tissues positive for COX-2 mRNA expression was much lower than that of tumor tissues. We also performed immunohistochemical staining to study the location of COX-2 in tumor tissues. Fig. 3 demonstrated that moderate to strong staining of COX-2 was predominantly detected in cancer cells, and the staining pattern was cytoplasmic. This staining was specific for COX-2 because immunoreactivity was lost when the antiserum to COX-2 was replaced with non-immune antiserum.
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Figure 1 Expression of COX-2 mRNA analyzed by RTPCR in normal and NPC tissues. T, tumor; N, normal.
Figure 2 Expression of COX-2 protein detected by western blot analysis in normal and NPC tissues. T, tumor; N, normal.
Discussion EBV infection is closely associated with NPC. One of the EBV genes that exhibits potent transforming activity is latent membrane protein 1 (LMP1). LMP1 is detected in at least 70% of NPC and all EBV-infected pre-invasive nasopharyngeal lesions, suggesting that LMP1 may play a role in NPC carcinoTable 2 Correlation between COX-2 expression and lymph node metastasis Stage
COX-2 expression
Stage
+
Total
N0 N1–N3
4 4
0 16
4 20
Statistical significance was evaluated by the Fisher’s exact test (P = 0.006).
Overexpression of COX-2 in NPC and association with lymph node metastasis
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7. Wilson KT, Fu S, Ramanujam KS, Meltzer SJ. Increased expression of inducible nitric oxide and COX-2 in Barrett’s esophagus and associated adenocarcinoma. Cancer Res 1998;58(14):2929–34. 8. Hida T, Yatabe Y, Achiwa H, et al. Increased expression of cyclooxygenase-2 occurs frequently in human lung cancers, specifically in adenocarcinoma. Cancer Res 1998;58(17): 3761–4. 9. Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, DuBois RN. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994;107(4):1183–8. 10. Liu CH, Chang SH, Narko K, et al. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001;276(21):18563–9. 11. Reddy BS, Rao CV, Seibert K. Evaluation of cyclooxygenase2 inhibitor for potential chemopreventive properties in colon carcinogenesis. Cancer Res 1996;56(20):4566–9. 12. Souza RF, Shewmake K, Beer DG, Cryer B, Spechler SJ. Selective inhibition of cyclooxygenase-2 suppresses growth and induces apoptosis in human esophageal adenocacinoma cells. Cancer Res 2000;60(20):5767–72. 13. Molina MA, Sitja-Arnau M, Lemoine MG, Frazier ML, Sinicrope FA. Increased cyclooxugase-2 expression in human pancreatic carcinoma and cell lines: growth inhibition by nonsteroidal anti-inflammatory drugs. Cancer Res 1999; 59(17):4356–62. 14. Tsujii M, Kawano S, Tsuji H, Sawaoka H, Hori M, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998;93(5):705–16. 15. Lee TH, Chuang LY, Hung WC. Induction of p21waf1 expression via Sp1-binding sites by tamoxifen in estrogen receptornegative lung cancer cells. Oncogene 2000;19(33): 3766–73. 16. Peng JP, Su CY, Chang HC, Chai CY, Hung WC. Overexpression of cyclooxygenase 2 in squamous cell carcinoma of the hypopharynx. Human Pathol 2002;33(1):1324– 1328. 17. Sheehan KM, Sheahan K, O’Donoghue DP, et al. The relationship between cyclooxygenase-2 expression and colorectal cancer. JAMA 1999;282(13):1254–7. 18. Uefuji K, Ichikura T, Mochizuki H. Cyclooxygenase-2 expression is related to prostaglandin biosynthesis and angiogenesis in human gastric cancer. Clin Cancer Res 2000;6(1): 135–8. 19. Shirahama T, Arima J, Akiba S, Sakakura C. Relation between cyclooxygenase-2 expression and tumor invasiveness and patient survival in transitional cell carcinoma of the urinary bladder. Cancer 2001;92(1):188–93. 20. Costa C, Soares R, Reis-Filho JS, Leitao D, Amendoeira I, Schmitt FC. Cyclooxygenase 2 expression is associated with angiogenesis and lymph node metastasis in human breast cancer. J Clin Pathol 2002;55(6):429–34. 21. Joki T, Heese O, Nikas DC, et al. Expression of cyclooxygenase-2 in human glioma and in vitro inhibition by a specific COX-2 inhibitor, NS398. Cancer Res 2000;60(17): 4926–31. 22. Tsao SW, Tramoutanis G, Dawson CW, Lo AK, Huang DP. The significance of LMP1 expression in nasopharyngeal carcinoma. Semin Cancer Biol 2002;12(6):473–87. 23. Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN. Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 1998;58(2): 362–6. 24. Mutoh M, Watanabe K, Kitamura T, et al. Involvement of prostaglandin E receptor subtype EP(4) in colon carcinogenesis. Cancer Res 2002;62(1):28–32.
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genesis.22 Recent study has demonstrated that LMP1 induces COX-2 expression in NPC cells. So, it is rational to speculate that COX-2 may be up-regulated in NPC tissues. Indeed, our data show that overexpression of COX-2 is frequently found in NPC tissues. On the contrary, the protein level of COX-2 in normal nasopharyngeal tissues is very low or undetectable. Overexpression of COX-2 provides several advantages for tumor growth progression. First, increased amount of PGs produced by COX-2 may stimulate tumor cell growth.23 Second, overexpression of COX-2 upregulates bcl-2 gene expression to prevent apoptosis of tumor cells.24 Moreover, COX2 may induce tumor angiogenesis and metastasis.25,26 Another important finding of this study is that we provide the first evidence that overexpression of COX-2 is closely linked with lymph node metastasis in NPC. Because NPC is characterized by its poor clinical outcome due to early metastasis and rapid progression, it will be important to study whether COX-2 can be developed as a prognostic factor for tumor metastasis in NPC patients. In agree with our results, a recent study showed that two specific COX-2 inhibitors, nimesulide and celecoxib, may induce growth arrest and apoptosis in various NPC cell lines. More importantly, nimesulide may suppress angiogenesis on chick choriollantoic membrane model.27 Collectively, our results suggest that expression of COX-2 is upregulated in NPC and selective COX-2 inhibitors may be useful for prevention or treatment of this malignancy.
Acknowledgement
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This study was supported by the grant CMRP8030 from Chang Gung University.
References
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1. Vokes EE, Liebowitz DN, Weichselbaum RR. Nasopharyngeal carcinoma. Lancet 1997;350(9084):1087–91. 2. Cheng SH, Jian JJ, Tsai SY, et al. Prognostic features and treatment outcome in locoregionally advanced nasopharyngeal carcinoma following concurrent chemotherapy and radiotherapy. Int J Radiat Oncol Biol Phys 1998;41(4): 755–62. 3. Cheng SH, Tsai SY, Yen KL, et al. Concomitant radiotherapy and chemotherapy for early-stage nasopharyngeal carcinoma. J Clin Oncol 2000;18(10):2040–5. 4. Dubois RN, Abramson SB, Crofford L, et al. Cyclooxygenase in biology and disease. FASEB J 1998;12(12):1063–73. 5. Goetzl E, An S, Smith WL. Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. FASEB J 1995;9(11):1051–8. 6. Ristimaki A, Honkanen N, Jankala H, Sipponen P, Harkonen M. Expression of cyclooxygenase-2 in human gastric carcinoma. Cancer Res 1997;57(7):1276–80.
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J.-P. Peng et al. in human gastric carcinoma. Am J Gastroenterol 1999; 94(2):451–5. 27. Chen PY, Long QC. Effects of cyclooxygenase 2 inhibitors on biological traits of nasopharyngeal carcinoma cells. Acta Pharmacol Sin 2004;25(7):943–9.
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25. Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA 1997;94(7):3336–40. 26. Murata H, Kawano S, Tsuji S, et al. Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis
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Overexpression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis
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Jyh-Ping Peng a, Hui-Chiu Chang b, Chung-Feng Hwang a, Wen-Chun Hung b,* a
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Department of Otolaryngology, Chang Gung University, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Kaohsiung, Taiwan, ROC b Institute of Biomedical Sciences, National Sun Yat-Sen University, No. 70, Lien-Hai Road, Kaohsiung 804, Taiwan, ROC Received 20 April 2005; accepted 4 May 2005
KEYWORDS
Summary Cyclooxygenase-2 (COX-2) has been shown to be involved in multiple steps of carcinogenesis. In this study, we examined COX-2 expression in nasopharyngeal carcinoma (NPC). COX-2 mRNA analyzed by reverse-transcription polymerase chain reaction (RT-PCR) was detected in 66% (16 of 24) of tumor tissues. Western blot analysis demonstrated that COX-2 protein level was increased in tumor tissues and was correlated with the expression level of mRNA. Immunohistochemical study showed that COX-2 was predominantly detected in cancer cells, and the staining pattern was cytoplasmic. Ten histologically normal nasopharyngeal tissues obtained from nasopharyngeal hyperplasia were also investigated. We found that COX-2 mRNA was detectable in three tissues and the COX-2 protein level was very low. The frequency of COX-2 overexpression was significantly higher in patients of the N1–N3 group than in patients of the N0 group (P = 0.006). Taken together, these data suggest that COX-2 is overexpressed and is associated with increased lymphatic invasion in NPC. c 2005 Elsevier Ltd. All rights reserved.
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Cyclooxygenase-2; Nasopharyngeal carcinoma; Metastasis; RT-PCT; Western blot analysis; Immunohistochemical staining
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Introduction
* Corresponding author. Tel.: +886 7 5252000; fax: +886 7 5250197. E-mail address: [email protected] (W.-C. Hung).
Nasopharyngeal carcinoma (NPC) is characterized by its poor clinical outcome due to early metastasis and rapid progression. There is a high prevalence of NPC in Southeast Asia, southern China, and the Mediterranean basin.1 In endemic areas such as
1368-8375/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2005.05.003
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microgram of total RNA was reverse-transcribed to complementary DNA (cDNA) by use of the OneStep reverse-transcription polymerase chain reaction (RT-PCR) kit (Qiagen) according to the manufacturer’s protocol. Each PCR reaction was performed in 50 lL of a reaction mix containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.4 mM deoxynucleotide triphosphate, 600 nM primers, and 3 U of HotStarTaq DNA polymerase. Five microliters of the reverse-transcribed cDNAs was added to the reaction mix and amplified for 30 cycles of denaturation at 94 C for 45 s, annealing at 60 C for 45 s, extension at 72 C for 2 min, and final extension at 72 C for 10 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. The primer sequence and PCR product size for COX-2, 50 -GGTCTGGTGCCTGGTCTGATGATG-30 (sense) and 50 -GTCCTTTCAAGGAATGGTGC-30 (antisense), 724 base pairs (bp). PCR product was run on 2% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light.
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Taiwan, 90% of these squamous cell carcinomas are WHO type IIa or IIb, that are very sensitive to radiation.2,3 Previous epidemiologic studies have indicated that several etiological factors including exposure of chemical carcinogens (such like Cantonese salted fish), and Epstein–Barr virus (EBV) infection may play an important role in the development of NPC. However, the mechanism by which these factors contribute to the tumorigenesis of NPC is largely unknown. Prostaglandins (PGs) are involved in the regulation of various physiologic and pathophysiologic processes, including blood clotting, kidney function, wound healing, cardiovascular disease, and inflammation.4 Recent works show that the conversion of arachidonic acid to PGs is catalized by cyclooxygenases (COXs). Two isoforms of COX, COX-1 and COX-2, have been identified. COX-1 is expressed constitutively in various tissues. Conversely, COX-2 expression is undetectable in most tissues and is induced by mitogens, cytokines, or tumor promoters.5 Lines of evidence suggest that COX-2 is participated in the steps of carcinogenesis. First, increased expression of COX-2 was frequently found in human cancers, including gastric, esophageal, lung, and colon cancer.6–9 Second, Overexpression of COX-2 is sufficient to induce tumorigenesis in transgenic mice.10 Third, non-steroidal anti-inflammatory drugs (NSAIDs), potent inhibitors of COXs, have been shown to exert chemopreventive effect on cancer development.11,12 More importantly, upregulation of COX-2 in cancer cells has been shown to link with increased angiogenesis and metastasis.13,14 In this study, we examine COX-2 expression in NPC and try to address its correlation with tumor metastasis.
Materials and methods
Normal and NPC samples
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Tumor tissues were obtained from 24 patients who underwent resection of tumors at Chang Gung Memorial Hospital, Kaohsiung Medical Center. Tumor tissues were sharply excised, placed immediately in liquid nitrogen, and stored at 80 C until use. Ten histologically normal hyperplasia tissues were also collected by the same method.
RNA extraction and reverse-transcription polymerase chain reaction Total RNA was isolated from tissues using a RNeasy Mini Kits from Qiagen (Santa Clarita, CA). One
Western blot analysis
Tissues were homogenized in a tissue homogenization buffer (50 mM Tris–HCl, pH 7.4, 250 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 2 mM sodium orthovanadate, 50 mM NaF, 1 mM phenylmethylsulphonyl fluoride, 1 mg/ml aprotinin, 2 lg/ml pepstatin A, and 2 lg/ml leupeptin). Tissue homogenates were centrifuged at 12,000g for 20 min at 4 C, and protein concentrations were determined using a BCA protein assay kit (Pierce, IL). Equal amounts of cellular proteins were subjected to separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and Western blot analysis was performed as described previously.15 Tumor tissues were classified as overexpression of protein when the signal intensity of tumor tissues was two-fold higher than the average intensity of the ten histologically normal hyperplasia tissues.
Immunohistochemistry Immunohistochemical staining was performed as described previously.16 In brief, 5-lm frozen sections were fixed with acetone and treated with 0.5% hydrogen peroxide to remove endogenous peroxidase activity. Tissue sections were probed with COX-2 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C. Tissue sections were washed twice with phosphate-buffered saline (PBS) and incubated with
Overexpression of COX-2 in NPC and association with lymph node metastasis
The expression of COX-2 in relation to lymph node metastasis was analyzed by using the Fisher’s exact test. A difference between groups of P < 0.05 was considered significant.
Results
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COX-2 is overexpressed in NPC The patients had a mean age 51 (range 32–72) years and included 18 men and 6 women. The degree of differentiation of the tumors and patient’s T and N status are summarized in Table 1. Of the 24 NPC tissues analyzed by RT-PCR,
Expression of COX-2 in nasopharyngeal carcinoma Sex
Stage
Differentiationa (WHO type)
COX-2 mRNA
COX-2 protein level
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
51 33 66 41 71 41 43 41 46 53 63 48 51 45 72 53 66 60 62 66 56 32 32 33
M M M F M F F M M F M F M M M F M M M M M M M M
T1N2 T4N0 T2N1 T2N1 T4N1 T2N1 T3N0 T2N3 T3N1 T1N2 T2N1 T3N2 T1N2 T1N2 T3N0 T4N0 T2N1 T2N3 T1N2 T3N1 T1N2 T3N1 T2N1 T1N1
IIb IIa IIa IIa I IIa IIa IIa IIa IIa IIb IIb I IIa IIa IIa IIa IIa IIa IIa IIa IIa IIa IIb
+ + + + + + + + + + + + + + + +
Overexpression Undetectable Overexpression Overexpression Overexpression Overexpression Undetectable Overexpression Overexpression Overexpression Overexpression Overexpression Overexpression Overexpression Undetectable Undetectable Undetectable Overexpressed Overexpressed Undetectable Overexpressed Overexpressed Undetectable Undetectable
Normal 1 2 3 4 5 6 7 8 9 10
+ + +
Undetectable Undetectable Undetectable Undetectable Low expression Low expression Low expression Undetectable Undetectable Undetectable
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Age (years)
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Table 1
Statistical analysis
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biotinylated secondary antibody (Dako, Copenhagen, Denmark) for 30 min at room temperature. The sections were washed with PBS, incubated with peroxidase-labeled streptavidin, and then developed by 3-amino-9-ethylcarbazole. Finally, the specimens were counterstained with Mayer hematoxylin and mounted with Entellan (Merck, Darmstadt, Germany). Negative controls were carried out by replacing primary antibody with non-immune antiserum. The 24 NPC tissues were categorized as WHO type and the T-stage distribution was classified according to the AJCC classification. From World Health Organization histological study, NPC samples of distinct histological types, including 2 squamous cell carcinoma (WHO type I), 18 non-keratinizing carcinoma (WHO type IIa), and 4 undifferentiated carcinoma (WHO type IIb) were studied.
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nasopharyngeal 47 24 27 31 54 36 39 57 65 16
tissues M M M M M F M F F M
Type I, squamous cell carcinoma; type IIa, non-keratinizing carcinoma; type IIb, undifferentiated carcinoma.
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Figure 3 Immunohistochemical analysis shows positive cytoplasmic staining for COX-2 protein in NPC tissue (Immunohistochemistry · 100).
COX-2 overexpression is associated with lymph node metastasis
Pathological investigations have demonstrated that COX-2 expression is associated lymph node metastasis in breast, colon, cervical, gastric and endometric cancers.17–21 Therefore, we addressed the correlation between COX-2 expression and lymph node metastasis in NPC patients. Table 2 showed that the frequency of COX-2 expression was significantly higher in the N1–N3 group than in the N0 group (P = 0.006, Fisher exact test). Additionally, all of the tumor tissues of the N0 group were negative for COX-2 mRNA expression. These results suggest that overexpression of COX-2 is associated with increased lymphatic invasion in NPC.
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expression of COX-2 mRNA was detected in 16 (66%) samples (Fig. 1 and Table 1). We also investigated histologically normal nasopharyngeal hyperplasia tissues. Our results demonstrated that low level of COX-2 mRNA was detected in three normal tissues. We next investigated COX-2 protein level in normal and tumor tissues by Western blot analysis and the autoradiograms were subjected to densitometric analysis. Tumor tissues were classified as overexpression of COX-2 protein when the signal intensity of tumor tissues was two-fold higher than the average signal intensity of 10 normal tissues. Our data demonstrated that COX-2 was highly expressed and was correlated with mRNA level in NPC tissues (Fig. 2). All of the NPC tumor tissues positive for COX-2 mRNA are found to be overexpression of COX-2 protein in Western blot analysis. Conversely, the COX-2 protein level of the three normal tissues positive for COX-2 mRNA expression was much lower than that of tumor tissues. We also performed immunohistochemical staining to study the location of COX-2 in tumor tissues. Fig. 3 demonstrated that moderate to strong staining of COX-2 was predominantly detected in cancer cells, and the staining pattern was cytoplasmic. This staining was specific for COX-2 because immunoreactivity was lost when the antiserum to COX-2 was replaced with non-immune antiserum.
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Figure 1 Expression of COX-2 mRNA analyzed by RTPCR in normal and NPC tissues. T, tumor; N, normal.
Figure 2 Expression of COX-2 protein detected by western blot analysis in normal and NPC tissues. T, tumor; N, normal.
Discussion EBV infection is closely associated with NPC. One of the EBV genes that exhibits potent transforming activity is latent membrane protein 1 (LMP1). LMP1 is detected in at least 70% of NPC and all EBV-infected pre-invasive nasopharyngeal lesions, suggesting that LMP1 may play a role in NPC carcinoTable 2 Correlation between COX-2 expression and lymph node metastasis Stage
COX-2 expression
Stage
+
Total
N0 N1–N3
4 4
0 16
4 20
Statistical significance was evaluated by the Fisher’s exact test (P = 0.006).
Overexpression of COX-2 in NPC and association with lymph node metastasis
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7. Wilson KT, Fu S, Ramanujam KS, Meltzer SJ. Increased expression of inducible nitric oxide and COX-2 in Barrett’s esophagus and associated adenocarcinoma. Cancer Res 1998;58(14):2929–34. 8. Hida T, Yatabe Y, Achiwa H, et al. Increased expression of cyclooxygenase-2 occurs frequently in human lung cancers, specifically in adenocarcinoma. Cancer Res 1998;58(17): 3761–4. 9. Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, DuBois RN. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994;107(4):1183–8. 10. Liu CH, Chang SH, Narko K, et al. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001;276(21):18563–9. 11. Reddy BS, Rao CV, Seibert K. Evaluation of cyclooxygenase2 inhibitor for potential chemopreventive properties in colon carcinogenesis. Cancer Res 1996;56(20):4566–9. 12. Souza RF, Shewmake K, Beer DG, Cryer B, Spechler SJ. Selective inhibition of cyclooxygenase-2 suppresses growth and induces apoptosis in human esophageal adenocacinoma cells. Cancer Res 2000;60(20):5767–72. 13. Molina MA, Sitja-Arnau M, Lemoine MG, Frazier ML, Sinicrope FA. Increased cyclooxugase-2 expression in human pancreatic carcinoma and cell lines: growth inhibition by nonsteroidal anti-inflammatory drugs. Cancer Res 1999; 59(17):4356–62. 14. Tsujii M, Kawano S, Tsuji H, Sawaoka H, Hori M, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998;93(5):705–16. 15. Lee TH, Chuang LY, Hung WC. Induction of p21waf1 expression via Sp1-binding sites by tamoxifen in estrogen receptornegative lung cancer cells. Oncogene 2000;19(33): 3766–73. 16. Peng JP, Su CY, Chang HC, Chai CY, Hung WC. Overexpression of cyclooxygenase 2 in squamous cell carcinoma of the hypopharynx. Human Pathol 2002;33(1):1324– 1328. 17. Sheehan KM, Sheahan K, O’Donoghue DP, et al. The relationship between cyclooxygenase-2 expression and colorectal cancer. JAMA 1999;282(13):1254–7. 18. Uefuji K, Ichikura T, Mochizuki H. Cyclooxygenase-2 expression is related to prostaglandin biosynthesis and angiogenesis in human gastric cancer. Clin Cancer Res 2000;6(1): 135–8. 19. Shirahama T, Arima J, Akiba S, Sakakura C. Relation between cyclooxygenase-2 expression and tumor invasiveness and patient survival in transitional cell carcinoma of the urinary bladder. Cancer 2001;92(1):188–93. 20. Costa C, Soares R, Reis-Filho JS, Leitao D, Amendoeira I, Schmitt FC. Cyclooxygenase 2 expression is associated with angiogenesis and lymph node metastasis in human breast cancer. J Clin Pathol 2002;55(6):429–34. 21. Joki T, Heese O, Nikas DC, et al. Expression of cyclooxygenase-2 in human glioma and in vitro inhibition by a specific COX-2 inhibitor, NS398. Cancer Res 2000;60(17): 4926–31. 22. Tsao SW, Tramoutanis G, Dawson CW, Lo AK, Huang DP. The significance of LMP1 expression in nasopharyngeal carcinoma. Semin Cancer Biol 2002;12(6):473–87. 23. Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN. Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 1998;58(2): 362–6. 24. Mutoh M, Watanabe K, Kitamura T, et al. Involvement of prostaglandin E receptor subtype EP(4) in colon carcinogenesis. Cancer Res 2002;62(1):28–32.
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genesis.22 Recent study has demonstrated that LMP1 induces COX-2 expression in NPC cells. So, it is rational to speculate that COX-2 may be up-regulated in NPC tissues. Indeed, our data show that overexpression of COX-2 is frequently found in NPC tissues. On the contrary, the protein level of COX-2 in normal nasopharyngeal tissues is very low or undetectable. Overexpression of COX-2 provides several advantages for tumor growth progression. First, increased amount of PGs produced by COX-2 may stimulate tumor cell growth.23 Second, overexpression of COX-2 upregulates bcl-2 gene expression to prevent apoptosis of tumor cells.24 Moreover, COX2 may induce tumor angiogenesis and metastasis.25,26 Another important finding of this study is that we provide the first evidence that overexpression of COX-2 is closely linked with lymph node metastasis in NPC. Because NPC is characterized by its poor clinical outcome due to early metastasis and rapid progression, it will be important to study whether COX-2 can be developed as a prognostic factor for tumor metastasis in NPC patients. In agree with our results, a recent study showed that two specific COX-2 inhibitors, nimesulide and celecoxib, may induce growth arrest and apoptosis in various NPC cell lines. More importantly, nimesulide may suppress angiogenesis on chick choriollantoic membrane model.27 Collectively, our results suggest that expression of COX-2 is upregulated in NPC and selective COX-2 inhibitors may be useful for prevention or treatment of this malignancy.
Acknowledgement
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This study was supported by the grant CMRP8030 from Chang Gung University.
References
R
1. Vokes EE, Liebowitz DN, Weichselbaum RR. Nasopharyngeal carcinoma. Lancet 1997;350(9084):1087–91. 2. Cheng SH, Jian JJ, Tsai SY, et al. Prognostic features and treatment outcome in locoregionally advanced nasopharyngeal carcinoma following concurrent chemotherapy and radiotherapy. Int J Radiat Oncol Biol Phys 1998;41(4): 755–62. 3. Cheng SH, Tsai SY, Yen KL, et al. Concomitant radiotherapy and chemotherapy for early-stage nasopharyngeal carcinoma. J Clin Oncol 2000;18(10):2040–5. 4. Dubois RN, Abramson SB, Crofford L, et al. Cyclooxygenase in biology and disease. FASEB J 1998;12(12):1063–73. 5. Goetzl E, An S, Smith WL. Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. FASEB J 1995;9(11):1051–8. 6. Ristimaki A, Honkanen N, Jankala H, Sipponen P, Harkonen M. Expression of cyclooxygenase-2 in human gastric carcinoma. Cancer Res 1997;57(7):1276–80.
907
908
J.-P. Peng et al. in human gastric carcinoma. Am J Gastroenterol 1999; 94(2):451–5. 27. Chen PY, Long QC. Effects of cyclooxygenase 2 inhibitors on biological traits of nasopharyngeal carcinoma cells. Acta Pharmacol Sin 2004;25(7):943–9.
R
ET
R
AC
TE
D
25. Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA 1997;94(7):3336–40. 26. Murata H, Kawano S, Tsuji S, et al. Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis