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Role of COX-2, Thromboxane A2 Synthase, and Prostaglandin I2 Synthase in Papillary Thyroid Carcinoma Growth

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Role of COX-2, Thromboxane A2 Synthase, and Prostaglandin I2 Synthase in Papillary Thyroid Carcinoma Growth Modern Pathology (2005) 18, 221–227 & 2005 USCAP, Inc All rights reserved 0893-3952/05 $30.00 www.modernpathology.org Role of COX-2, thromboxane A2 synthase, and prostaglandin I2 synthase in papillary thyroid carcinoma growth Sabine Kajita, Katharina H Ruebel, Mary B Casey, Nobuki Nakamura and Ricardo V Lloyd Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA The development of papillary thyroid carcinoma is influenced by many factors including genetic alterations, growth factors, and physical agents such as radiation. Arachidonic acid and its derivatives including prostaglandins (PG) and thromboxane along with the enzymes involved in their synthesis have been shown to influence the growth of various tumors. We analyzed the immunoreactivity for cyclooxygenase-2 (COX-2) and mRNA expression levels of the enzymes COX-2, thromboxane A2 (TXA2) synthase, and PGI2 synthase by RT- PCR in papillary carcinomas and matching normal tissues to determine the role of these enzymes in the development of papillary thyroid carcinomas. A papillary thyroid carcinoma cell line TPC-1 was also studied in vitro to determine the role of the specific COX-2 inhibitor NS-398 on COX-2 and vascular endothelial growth factor-A, since COX-2 also has a role in regulating tumor angiogenesis. RT-PCR analysis showed significant increases in TXA2 synthase mRNA levels in papillary thyroid carcinomas compared to normal thyroid tissues. Although COX-2 mRNA levels were generally increased in papillary carcinomas, the differences were not statistically significant. There were no significant differences in PGI2 synthase mRNA levels. COX-2 protein expression was greater in papillary carcinoma compared to normal thyroid tissues; however, the levels were quite variable. In vitro studies with a COX-2 inhibitor, NS-398, showed inhibition of tumor growth along with increased levels of COX-2 and vascular endothelial growth factor-A mRNA expression. These results indicate that specific enzyme levels in the PG synthesis pathway such as TXA2 synthase are increased in papillary thyroid carcinomas. COX-2 also has a role in papillary thyroid growth, since a specific inhibitor of COX-2 regulates papillary thyroid carcinoma cell proliferation. These results implicate several enzymes in the synthesis of prostanoids as regulators of thyroid papillary carcinoma proliferation and suggest that increased levels of expression of these enzymes may play a role in the pathogenesis of these tumors. Modern Pathology (2005) 18, 221–227, advance online publication, 8 October 2004; doi:10.1038/modpathol.3800285 Keywords: COX-2; thyroid; papillary carcinoma; thromboxane synthase Arachidonic acid and its derivatives, the prosta- inflammatory drugs (NSAIDs) or by selective COX-2 glandins and thromboxane (Figure 1), are important inhibitors.7–10 in many physiological processes.1 They are also COX-2 is expressed in many types of cancers mediators of tumor progression. There are two major including colon,11,12 pancreas,8,13,14 stomach,15–17 cyclooxygenases (COX). COX-1 is the constitutive lung,12,18 breast,12,19 prostate,20 cervix,6 head and form of the enzyme, which catalyzes formation of neck,21 esophagus,22 bladder,23 glioma,24 and mela- prostaglandin from arachidonic acid, while COX-2 nomas.25 Recent studies have shown that COX-2 is is expressed as an inducible isoform. COX-2 expres- expressed in thyroid lesions.26–29 Thyroid tumors sion is increased by various substances including generally express higher levels of COX-2 than mitogens,2 tumor promoters,3,4 cytokines,1,2,5,6 ser- normal tissues and thyroid tissues from patients um,5 and free fatty acids.7 Other studies have shown with thyroiditis.26–29 that COX-2 is also regulated by nonsteroidal anti- Thromboxane A2 (TXA2) synthase and prosta- glandin (PG) I2 synthase are enzymes located down- stream from COX-1 and COX-2, and catalyze Correspondence: Dr RV Lloyd, MD, PhD, Department of synthesis of PG H, TXA2 and PGI2 (Figure 1). TXA2 Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street, stimulates platelet aggregation, while PGI2 inhibits SW, Rochester, MN 55905, USA. aggregation of platelets. Some studies have sug- E-mail: [email protected] Received 22 June 2004; revised 16 August 2004; accepted 17 gested that TXA2 synthase and PGI2 synthase 30–32 August 2004; published online 8 October 2004 contribute to tumor metastasis. TXA2 synthase Role of COX-2, TXA2, and PGI2 S Kajita et al 222 was shown to stimulate tumor angiogenesis, while and three cases of follicular variant of papillary 33–35 PGI2 synthase inhibited angiogenesis. Our re- carcinomas. cent studies on COX-2 and TXA2 synthase expres- Immunostaining for COX-2 with a monoclonal sion in thyroid tumors and other thyroid lesions antibody from Cayman Chemicals, Ann Arbor, MI at using immunohistochemistry found higher levels of a 1/200 dilution was carried out using frozen tissues these proteins in papillary thyroid carcinomas fixed in formalin.36 compared to adenomatous nodules and benign thyroid tumors.36 The present study examines COX-2, TXA2 synthase, and PGI2 synthase mRNA RNA Extraction and RT-PCR expression in normal thyroid tissue and papillary thyroid carcinomas using semiquantitative RT-PCR Total RNA was extracted from frozen normal and tumor tissue samples and from cultured cells using to analyze the role of these enzymes in thyroid 37 tumor growth. We also examined the selective the TRIzol reagent as previously reported. A inhibition of COX-2 by NS-398 in the papillary measure of 1 mg total RNA was converted to cDNA thyroid carcinoma cell line TPC-1 to study the using the Prostar First Strand RT-PCR Kit (Strata- effects of this inhibitor on cell growth as well as gene, La Jolla, CA, USA) according to the manufac- COX-2 and VEGF-A mRNA expression. turer’s instruction. A measure of 5 ml cDNA was amplified by hot-start PCR in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). Five specific primer sets with individual Materials and methods annealing temperatures were used (Table 1). Each PCR reaction was performed using 1 Â PCR buffer, Tissue Samples 2 mM MgCl2, 1.25 U Taq polymerase (Promega, In all, 15 cases of papillary thyroid carcinomas and Madision, WI, USA), 0.2 mM each dNTP (Roche adjacent normal tissues were obtained at the time of Diagnostics, Alameda, CA, USA) in a final volume of surgery and stored at À701C. Hematoxylin and eosin 50 ml. PCR product identity was verified by auto- staining was performed for each case to determine mated sequencing using the 3730XL DNA Analyzer the diagnosis and distribution of tumor and normal (Applied Biosystems, Foster City, CA, USA). In all, thyroid cells. There were 12 cases of classic variant 20 ml of each PCR reaction as analyzed on a 1.5% agarose gel, stained briefly in ethidium bromide, and viewed under UV light. Densitometric Analysis Densitometric analysis was carried out with a Quantity One System (Bio-Rad Laboratories, Her- cules, CA, USA). Relative densities of the samples were calculated and compared with the house- keeping gene HPRT expression level. Samples were normalized and expressed relative to HPRT. Figure 1 Schematic diagram showing the metabolic pathways of Cell Culture prostanoids. Arachidonic acid is converted to PGH2 under the influence of COX-1 and COX-2. TXA2 synthase influences A human papillary thyroid carcinoma cell line, synthesis of TXA2 while PGI2 synthase influences PGI2 formation. TPC1, was obtained from Dr Yuri Nikiforov (Uni- Table 1 Sequence of primers used in experiments Sequence Cycle Product Annealing References number (bp) temperature (1C) HPRT Forward 50-CTTGCTCGAGATGTGATGAAG-30 28 290 58 38 Reverse 50-GTCTGCATTGTTTTGCCAGTG-30 COX-2 Forward 50-GGTCTGGTGCCTGGTCTCATC-30 30 724 56 39 Reverse 50-GTCCTTTCAAGGAGAATGGTGC-30 0 0 40 TXA2 synthase Forward 5 -CCTTCTCCTGGCTCATTTA-3 32 270 56 Reverse 50-TCGTCTCGGTTCTTATTGG-30 0 0 41 PGI2 synthase Forward 5 -AGGAGAAGCACGGTGACATC-3 35 387 65 Reverse 50-GCAGCGCCTCAATTCCGTAA-30 VEGF-A 165 Forward 50-AGGAGGAGGGCAGAATCATCA-30 30 336 56 42 Reverse 50-CAGGGATTTTCTTGTCTTG-30 Modern Pathology (2005) 18, 221–227 Role of COX-2, TXA2, and PGI2 S Kajita et al 223 versity of Cincinnati Medical Center). The cell line Table 2 Distribution of cases of papillary thyroid carcinomas has a rearranged form of RET/PTC1.43 Cells were cultured in DMEM, supplemented with 10% FBS, Case Age/ Tumor Extra- Lymph node COX-2 number sex size thyroidal metastasis immunoreactivity 1 mg/ml insulin, 100 U/ml penicillin, 100 mg/ml (cm) extenasion streptomycin, and 0.25 mg/ml fungizone (InVitrogen Normal Tumor Life Technologies, Carlsbad, CA, USA) using cell 44 culture conditions as previously reported. 1 68/M 3 + + 0 2+ 2 35/F 3 ÀÀ1+ 3+ 3 59/F 3.5 + + 1+ 2+ Cell Treatments and Reagents 4 77/M 3.5 + + 1+ 1+ 5 70/M 3.2 ÀÀ1+ 3+ 6 52/M 1.8 À +1+1+ A stock solution of 40 mM NS398 (Cayman Chemi- 7 46/F 0.6 ÀÀ1+ 1+ cal Company, Ann Arbor, MI, USA) was dissolved in 8 22/F 3.5 À +1+2+ dimethyl sulfoxide (DMSO) (Sigma) and diluted in 9 43/F 4 ÀÀ02+ standard growth medium to a final concentration of 10 41/M 3.5 ÀÀ02+ 20–80 mM for the cell growth experiments. COX-2 11 52/M 5 + + 1+ 1+ 12 59/F 5.2 ÀÀ1+ 3+ expression experiments were performed with NS- 13 31/M 6 À +1+1+ 398, which was diluted in serum-free medium and 14 46/M 2.5 ÀÀ1+ 1+ treated with 80 mM NS-398. The final concentration 15 62/F 5.3 + À 1+ 1+ of DMSO was 0.2% in each experiment. The experiments were performed at least three times.
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