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Endocrinology, doi:10.1210/en.2003-0915
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Endocrinology Vol. 145, No. 5 2561-2571
Copyright © 2004 by The Endocrine Society

Tumor Growth Inhibition by Indomethacin in a Mouse Model of Human Medullary Thyroid Cancer: Implication of Cyclooxygenases and 15-Hydroxyprostaglandin Dehydrogenase

Virginie Quidville, Nadine Segond, Elisabeth Pidoux, Regis Cohen, Annick Jullienne and Sylvie Lausson

Institut National de la Santé et de la Recherche Médicale, Unité 349, Hôpital Lariboisière, 75475 Paris Cedex 10, France

Address all correspondence and requests for reprints to: Dr. S. Lausson, Institut National de la Santé et de la Recherche Médicale Unité 349, Hôpital Lariboisière, Centre Viggo Petersen, 2 rue Ambroise Paré, 75475 Paris Cedex 10, France. E-mail: sylvielausson{at}aol.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Medullary thyroid cancer (MTC) is a C cell neoplasm-secreting calcitonin. Surgery remains the only treatment as the primary tumor and metastases resist radio- and chemotherapies. MTC produces high amounts of prostaglandins (PGs). Nonsteroidal antiinflammatory drugs have an antitumoral effect, generally related to the decrease of PG levels. We assessed the therapeutic potential of indomethacin in a model of human (TT cells) tumors in nude mice. Indomethacin (1.5 or 2.0 mg/kg body weight·d for 7 wk) inhibited tumor volume by 49 or 77%, respectively, and decreased the plasma level of CT. Although the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling method revealed few apoptotic nuclei, the number of proliferating cells was significantly decreased (Ki-67 antigen study). Immunological effector recruitment and vascular network was not modified by treatment. The inducible synthesis enzyme, cyclooxygenase-2 (COX-2), was revealed only in infiltrating cells, both in treated and control tumors. The expression of the constitutive synthesis enzyme COX-1 was diminished, and the expression of 15-prostaglandin dehydrogenase, the key enzyme catabolizing PGs, was increased in treated tumors. Thus, our results demonstrated the potential of indomethacin, inhibitor of COX-1 and COX-2, to prevent MTC growth. The synthesis enzyme, COX-1, and the catabolism enzyme 15-prostaglandin dehydrogenase, could be involved in MTC development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEDULLARY THYROID CARCINOMA (MTC) is an endocrine neoplasm of C cells (for review, see Ref. 1). This thyroid tumor can be sporadic in about 75% of cases; the remaining 25% are represented by familial forms (2). Hereditary germ-line mutations or somatic mutations of the RET proto-oncogene are involved in the carcinogenesis of familial or sporadic MTCs (3, 4). MTC synthesizes and secretes large amounts of calcitonin (CT) (5) and other molecules, in particular CT gene-related peptide and carcinoembryonic antigen (1). CT detected in plasma or by immunohistochemistry is the most specific marker for MTC (6). Plasma CT is generally correlated to tumor size (7).

The clinical course of patients with MTC is variable, ranging from indolent to extremely aggressive (8). Patients with sporadic carcinoma are commonly diagnosed at a late stage. Treatment effectiveness is largely related to the stage of the disease at the time of the diagnosis. The only treatment of MTC is the surgical removal of neoplasic tissue. MTC is currently managed by total thyroidectomy and microdissection of cervicomediastinal lymph nodes. Conventional radiotherapies and chemotherapies play a marginal role in advanced MTC (1).

Thus, new chemopreventive approaches using nonsteroidal antiinflammatory drugs (NSAIDs) could be effective in MTC treatment. Evidence has been accumulated from investigations in animals and humans that NSAIDs retard the development of colon (9, 10, 11, 12, 13), gastrointestinal (14), prostate (15), breast, and lung cancers (16). Prostaglandins (PGs) have a well-established role on tumorigenicity (17). NSAIDs inhibit PG synthesis enzymes, cyclooxygenases 1 and 2 (COX-1 and COX-2). Although COX-1 is constitutively expressed in most tissues, COX-2 is usually absent in most tissues, but can be induced by physiological and pathological stimuli. COX-2 is overexpressed in pancreatic (18, 19), lung (20, 21), breast (22), colon (23, 24), and prostate cancers (25). Moreover, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme of PG catabolism, is decreased in high-grade bladder tumors (26). Furthermore, Rao et al. (27) suggest that inhibition of 15-PGDH activity plays a role in the enhancement by genistein of experimental murine tumors of colon.

The antitumoral effects of NSAIDs are linked to an inhibition of tumor cell division and/or tumor cell death by apoptosis (28, 29, 30, 31, 32). NSAIDs can also act on tumorigenicity by enhancement of the immune response (33, 17) and/or by inhibiting angiogenesis (34, 35, 36, 37).

Although Williams et al. (38) found, in 1968, high PG levels in plasma and tumor tissues of patients with MTC, the antitumor potential of NSAIDs was never tested in this cancer. The in vitro model of human MTC is the TT cell line. These cells produce high levels of CT (39) and express CT receptor, suggesting the existence of an autocrine/paracrine control of proliferation by CT (40). We previously reported that TT cells and MTC expressed 15-PGDH mRNA and protein (41). Moreover, the well-known classical NSAID, indomethacin, a COX-1 and COX-2 inhibitor, decreased TT cell proliferation and increased 15-PGDH levels and activity. The 15-PGDH activity was negatively correlated with TT cell proliferation (42).

The aim of this study was to establish the therapeutic potential of NSAIDs for MTC and to investigate the immunological and/or vascular and enzymatic mechanisms involved. Our results showed that indomethacin inhibited the development of provoked TT tumors in athymic mice. This NSAID acts on tumor cell division without affecting cell viability. Angiogenesis and immune effector recruitment were not modified by treatment. The increase of 15-PGDH expression and the decrease of COX-1 expression in treated tumors suggested the implication of these enzymes in the indomethacin effect.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The TT human cell line was supplied by American Type Culture Collection (Rockville, MD). This cell line was routinely maintained in RPMI 1640 medium (Invitrogen Life Technologies, Cergy Pontoise, France) supplemented with heat-inactived fetal calf serum (FCS; 10%), L-glutamine (6 mM), and HEPES (10 mM). The medium was changed every 2 d. For each experiment, cells were dissociated with trypsin-EDTA and counted in an hemocytometer. In vitro experiments were repeated three times, and each point is the mean of three replicates except for Ki-67 studies, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL), and caspase activity (four replicates).

Reversibility studies
TT cells were seeded out at 150,000 cells/well in six-well plates. Three replicates were used at each time for each treatment. After 48-h cell adhesion, the cells were cycled by culture in a medium without FCS for 30 h. From this time, considered as d 0, all the cells were cultured in normal medium containing 10% FCS, until the end of the experiments. At d 0, indomethacin (Cayman Chemical, Ann Arbor, MI), dissolved in carbonate buffer (0.2 M sodium carbonate buffered to pH 7.5 with 0.2 M monosodium phosphate), was added to cell culture medium at 200 µM final concentration. In parallel, control cells were cultured in normal medium without indomethacin, from d 0 to d 12. At d 6, cells in some wells were dissociated with trypsin-EDTA and counted. Indomethacin treatment was stopped in half of the other wells and replaced by normal medium. In the other half, the treatment was continued during next 6 d. At d 12, cells were dissociated with trypsin-EDTA, and the number of cells was determined. The cell viability was evaluated using the trypan blue exclusion test. Finally, TT cells were centrifuged, the pellets were frozen at –80 C until Western blot analysis and 15-PGDH activity assay.

Prostaglandin E2 (PGE2) determination
Dimethyl PGE2 (dmPGE2) treatment.
TT cells were seeded out and synchronized as described above. Indomethacin (200 µM) and/or 10 µM of dmPGE2 (Cayman Chemical), stable PGE2 analog (neither degraded by 15-PGDH nor recognized by the antibody used in the ELISA), were added to the cell culture medium for 48 h. Cells were dissociated with trypsin-EDTA and counted. After centrifugation, the pellets were frozen at –80 C until PGE2 and 15-PGDH activity assays.

PGE2 assay.
The assay was performed on 2 µl of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate cellular lysate (2 x 104 cells). The intracellular PGE2 levels were measured using a commercial EIA kit (Cayman Chemical) according to the manufacturer’s instructions.

DNA synthesis
The indomethacin effect on DNA synthesis in TT cells was investigated using [3H]thymidine incorporation. TT cells were seeded out at 75,000 cells/well in 12-well plates. The [3H]thymidine (specific activity: 666 GBq/mmol) was added to the medium (18.5 MBq/well) at the same time as indomethacin for 6 h.

Animals and tumor cell inoculation
Athymic female mice (Swiss nu/nu, 8 wk old, 20 g) purchased from IFFA CREDO (L’Arbresle, France) were maintained in our animal facilities under pathogen-free conditions. Mice had continuous free access to sterilized food and autoclaved water. Experiments were started after 1 wk of acclimatization. For tumor cell inoculation, subconfluent TT cells were dissociated with trypsin-EDTA and suspended in PBS at a density of 5 x 107 cells/ml. The TT cell suspension was inoculated (5 x 106 cells/animal) sc on the back of each mouse at d 0. Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators.

Tumor growth in nude mice and indomethacin administration
The animals were randomly divided after inoculation of tumor cells into three groups: one control group (n = 8) and two groups receiving indomethacin (1.5 mg/kg body weight·d, n = 8; or 2 mg/kg·d, n = 6, respectively) during 7 wk in drinking water. For initial solution, indomethacin was dissolved in ethanol at 9 mg/ml concentration. To evaluate the ingested quantity of indomethacin, the drinking water was recorded every 2 d. When tumors became palpable, their diameters were measured with a caliper each week and tumor volume (mm3) was calculated using the following formula: (the shortest diameter)2 x (the longest diameter) x 0.5. There was no difference in body weight among the groups throughout the experiment.

Histology of tumors
After 7 wk, all tumors (localized under the skin) were excised, weighed, and divided in two parts. One part was embedded in cryocompound Tissue-Tek (Sakura Finetek Europe BV, Zoeterwoude, The Netherlands) and immediately frozen in –40 C cooled isopentane. For immunohistochemical studies, tissue sections (10 µm) were fixed in 4% paraformaldehyde in PBS. For morphological studies involving Mann Dominici staining, the other part of tumor was previously fixed in 4% paraformaldehyde in PBS during 36 h, incubated in 15% sucrose in PBS overnight at +4 C, embedded in cryocompound Tissue-Tek, and then frozen in –40 C cooled isopentane.

CT determination
CT determination was performed in mouse plasma and TT cell culture medium. Blood was obtained by intracardiac puncture from anesthetized mice, before the animals were killed. TT cells were treated or not treated with 200 or 400 µM indomethacin. Culture medium was collected at d 2, 4, and 6. CT level was assayed in plasma and culture medium by RIA using antibodies specific for human CT (hCT) as previously described (43). In brief, 10 µl of plasma were incubated with antibody against human CT, at 1/400,000 in phosphate buffer (sheep, Institut National de la Recherche Agronomique, Dr. Barlet, Clermont Ferrand, France) during 4 d. One hundred microliters of hCT labeled with 125I (125I-tyrosyl-CT, Amersham Pharmacia Biotech, Saclay, France) were added and incubated during 4 more days. Free 125I hCT was separated from bound hCT by adsorption on dextran-charcoal. Radioactivity was measured by gamma counting.

Protein extraction
Proteins from TT cell pellet and tumor samples were extracted using a lysis buffer containing 16 mM CHAPS (Sigma, St Quentin Fallavier, France), 20 mM Tris-HCl (pH 7.5), 1 mM Na2EDTA, 1 mM dithiothreitol (DTT), protease inhibitor cocktail (Sigma), and 0.5 mM phenylmethylsulfonylfluoride. Samples were sonicated and then centrifuged at 15,000 x g for 20 min and supernatants collected. The protein content was measured with a protein assay kit (Bio-Rad, Ivry sur Seine, France) according to the manufacturer’s instructions.

15-PGDH activity assay
The detection of the 15-PGDH activity in cell lysates was based on the stereospecific transfer of 15-[3H] tritium from 5,6,8,11,12,14,15-3H(N)-PGE2 to glutamate by coupling 15-PGDH with glutamate deshydrogenase (44). The reaction mixture contained: NH4Cl (5 mM), monosodium {alpha}-ketoglutarate (1 mM), oxidized nicotinamide-adenine dinucleotide (1 mM) (Sigma), DTT (1 µM), 5,6,8,11,12,14,15-3H(N)-PGE2 (1 µM, 200 Ci/mmol) (PerkinElmer Life Sciences, Courtabeuf, France), glutamate deshydrogenase (50 µg) (Roche Diagnostics, Meylan, France), and appropriate amount of cellular lysate containing 15-PGDH, in a final volume of 1 ml of Tris-HCl buffer (50 mM, pH 7.5). The reaction was carried out for 12 min at +37 C and stopped by the addition of 0.3 ml of aqueous charcoal suspension (10% charcoal in 1% dextran solution). The reaction mixture was then centrifuged at 3000 x g for 15 min at +4 C after standing for 5 min at room temperature. The supernatant containing [3H]glutamate was collected and radioactivity was determined by liquid scintillation counting.

Western blot analysis of 15-PGDH, COX-1, and COX-2 protein expression
Proteins extracted from cell or tumor samples (30 and 80 µg for COX-1 and COX-2, respectively) were separated electrophoretically on a 4–20% SDS-Tris-glycin polyacrylamide gel (NOVEX, Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (PerkinElmer Life Sciences). Transfer efficiency was visualized using prestained protein standard (SeeBlue, NOVEX, Invitrogen).

Membranes were blocked overnight at +4 C in Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dried milk. The primary antibodies, anti-15-PGDH: 1/10,000 (generous gift from Professor Taï, University of Kentucky, Lexington, KY) and anti-actin: 1/300 (Sigma), were incubated with the membranes for 1 h at room temperature. Then the membranes were washed extensively and reincubated with peroxidase-conjugated secondary antibody antirabbit IgG (Sigma) for 1 h at a final dilution of 1/15,000. ß-Actin was revealed following the same protocol. For COX-1, the primary antibody diluted at 1/200 (Santa Cruz Biotechnology, Heidelberg, Germany) was incubated with the membrane overnight at +4 C. For COX-2, the primary antibody diluted at 1/500 (Santa Cruz Biotechnology) was incubated with the membrane for 2 h at room temperature. After washes, the membrane was reincubated with peroxidase-conjugated secondary antibody antigoat IgG (Santa Cruz Biotechnology) for 1 h at 1/10,000. After washes, the ECL Detection System (Amersham Pharmacia Biotech) was used to generate specific signal. Then, the bands were quantified using Bio-1D software (Vilber Lourmat, Marne la Vallée, France) and the 15-PGDH or COX-1 content was standardized using actin density.

Immunohistochemistry studies in tumor tissues
15-PGDH and COX-1 protein detection.
Frozen tissue sections (10 µm) were fixed in cold acetone (–20 C) during 10 min just before staining and then washed with PBS. Nonspecific binding sites were blocked with PBS 4% albumin during 30 min at room temperature in a moist chamber. Then, slices were incubated overnight at +4 C with anti-15-PGDH polyclonal antibody diluted to 1/100 or with anti-COX-1 polyclonal antibody (Cayman Chemical) diluted to 1/50 in PBS 1% albumin. Negative controls were performed in the absence of primary specific antibody. After washing with PBS 0.5% Triton X-100 for 20 min, and then washing with PBS for 20 min, slides were incubated for 2 h at room temperature in a moist chamber with fluorescein-conjugated secondary antibody against rabbit IgG (Sigma) diluted to 1/200 in PBS 1% albumin. Slices were rinsed with PBS 0.5% Triton X-100 and mounted in fluorescent mounting medium (Dako, Trappes, France).

Macrophage detection.
Following the same protocol, macrophages were detected after slice incubation with antibody antimouse CD11b-R-phycoerythrin (Interchim, Leinco Technologies, Inc., St. Louis, MO) diluted to 1/50 (45).

COX-2 detection.
Immunostaining was performed by the avidin-biotin complex technique using the Vectastain ABC-AP kit (Vector Laboratories, Burlingame, CA). Briefly, cryostat sections were fixed in 4% paraformaldehyde. Nonspecific binding sites were blocked with the normal serum from the kit for 10 min. The primary goat polyclonal specific antibody against COX-2 (Santa Cruz) was diluted 1/400 and incubated overnight at room temperature. Sections were then incubated with a 1/200 dilution of biotinylated rabbit antigoat IgG for 30 min at room temperature. After PBS washes, sections were incubated with ABC-AP reagent (Vector Laboratories) for 30 min at room temperature. Sections were rinsed and incubated in alkaline phosphatase substrate solution (Vector Red, Vector Laboratories). Finally, the sections were counterstained in hematoxylin. Formalin-fixed, paraffin-embedded sections of human colorectal carcinomas known to have strong immunolabeling for COX-2 were used as positive control.

Angiogenesis in tumor tissues.
Frozen tissue sections were fixed in 4% paraformaldehyde. We then followed the protocol described for 15-PGDH and COX-1 detection. To analyze the presence of vessels in tumors, tissue sections were incubated with antifactor VIII-related antigen antibody (Dako) diluted to 1/200.

Apoptosis detection
TUNEL method.
The DNA strand breaks characteristic of apoptosis in TT cells grown in Lab-Tek Chamber Slides and tissue sections were revealed by the In Situ Cell Death Detection Kit (Roche Diagnostics). Terminal deoxynucleotidyl-transferase catalyzes polymerization of fluorescein-labeled nucleotides to free 3'OH DNA ends. Labeled nuclei were observed by fluorescence microscopy.

Caspase-3 activity determination.
TT cells (150,000 cells/well) were treated for 48 h with indomethacin at 100 and 200 µM in six-well plates containing culture medium with 1% fetal bovine serum. Four replicates were used for each treatment. Culture medium was collected, cells were dissociated with trypsin-EDTA, added to the corresponding medium, and then centrifuged for 5 min at 15,000 rpm. After one wash with PBS at +4 C and centrifugation, cell pellets were lysated during 20 min in 150 µl of lysis buffer containing 10 mM pH 7.4 Tris-HCl, 200 mM NaCl, 5 mM EDTA, 10% glycerol, and 1% Nonidet P-40. Caspase-3 activity was determined by the cleavage of specific caspase-3 substrate composed of a tetrapeptide, DEVD (Asp-Glu-Val-Asp), coupled with fluorophore, AFC (7-amino-4 trifluoromethylcoumarin). The reaction mixture contained 100 µl of cellular lysate, 200 µl of reaction buffer (0.1 mM phenylmethylsulfonylfluoride, 10 mM DTT, and 10 mM HEPES, pH 7.4) and 10 µl of DEVD-AFC substrate (Biosource, Nivelles, Belgium). After incubation for 2 h at 37 C in the dark, the light emitted by AFC liberated was quantified by a spectrofluorometer ({lambda}ex = 400 nm, {lambda}em = 505 nm). Simultaneously, the proteins were estimated in each sample by a DC protein assay kit (Bio-Rad). Results of caspase-3 activity assay were corrected by the protein content of each sample.

Mitotic index determination
The number of nuclei was estimated using the Ki-67 nuclear protein detection (46) by immunofluorescence as described above. TT cells grown in Lab-Tek Chamber Slides, and tissue sections fixed respectively in 4% paraformaldehyde or cold acetone, were incubated with primary antibody against Ki-67 protein (Zymed, Clinisciences, Montrouge, France) at 1/50 dilution overnight at +4 C. The specimens were treated with fluorescein-conjugated secondary antibody antirabbit IgG (Sigma) at 1/200 dilution. The number of Ki-67-positive cells was counted in more than 20 areas of 0.127 mm2. After 4',6-diamidino-2-phenylindole coloration of adjacent slides, total nuclei were assessed, and the mitotic index was calculated as followed: mitotic index (%) = (number of cells labeled with Ki-67/total cell number) x 100.

Statistical analysis
Data are expressed as mean ± SE. In vitro data, volumes and weights of tumors, and CT levels were compared by one-way ANOVA at each time followed by Fisher tests. Variations of the mitotic index, angiogenesis, and levels of 15-PGDH, and COX-1 proteins were examined by Student’s t tests. The relationships between 15-PGDH activity and the number of cells (in percent of control at each day) and that between CT levels and cell number per well were tested by simple linear regression. The relationship between plasma CT and tumor weight in control mice was also studied by simple linear regression.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of indomethacin on TT cell culture
Cellular proliferation and cell death.
Indomethacin (200 µM) induced a significant inhibition of TT cell growth (P < 0.0001; Fig. 1AGo). The cell numbers were 44–49% of controls after 6, 8, 10, and 12 d of treatment. When indomethacin treatment was removed, the cell number increased significantly without reaching control levels. Only the high dose of indomethacin (200 µM during 2 d) significantly enhanced cell death as assessed by two different methods. TUNEL method revealed an increase of labeled nuclei number (26 ± 1.9% vs. 8.9 ± 2.7% in controls; P < 0.05), and caspase-3 activity levels increased significantly (60.4 ± 6.3 arbitrary units per µg protein vs. 4.9 ± 0.3 in controls; P < 0.0001). These experiments were performed, as indicated in the protocols, in cells cultured in medium containing 1% FCS. When TUNEL determination was realized on cells maintained in 10% FCS, labeled nuclei number was not significantly increased (9.4 ± 0.3% vs. 7.2 ± 5.9% in controls). We also assessed indomethacin effect on cell division following [3H]thymidine incorporation after 6-h exposure to various concentrations of indomethacin. Figure 2Go shows a 28% significant decrease of DNA synthesis activity at 100 µM. The effect was more important at 200 µM (48%). Thus, indomethacin had a rapid effect on DNA synthesis in TT cells, which could explain the decrease of cell proliferation. Ki-67 mitotic index was decreased by 200 µM indomethacin treatment (32.2 ± 1.8% of labeled nuclei in controls vs. 0 in treated cells) indicating that TT cells were arrested in the G0 phase.



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  		FIG. 1. A, Reversible effect of indomethacin on TT cell proliferation. Cells were seeded out at 150,000 cells/well and treated (indo +) or untreated (control) for 12 d with 200 µM of indomethacin. For reversibility studies, indomethacin was suppressed from d 6–12 in a part of cultures (indo-) and replaced by normal culture medium. Cells were counted and harvested as described in Materials and Methods. Indomethacin decreased TT cells growth significantly at 6, 8, 10, and 12 d of treatment. The cell proliferation is expressed as the number of cells/well. B, Reversible effect of indomethacin treatment on 15-PGDH activity in TT cells. Proteins of the cell pellets obtained were extracted using a CHAPS lysis buffer. The 15-PGDH activity measure was performed by the enzymatic coupling method of Taï (44 ). Indomethacin (indo +) increased significantly the 15-PGDH activity at 6, 8, 10, and 12 d of treatment. This effect was reversible when indomethacin was suppressed (indo-) in the culture medium. Panels A and B are representative graphs of three independent experiments performed in triplicates. ANOVA and Fisher tests were performed each time (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. indo +). C, 15-PGDH protein expression in TT cells. Cellular lysate protein (20 µg/lane) was loaded on a SDS-polyacrylamide gel (4–20%), electroblotted, and transferred on a PVDF membrane. The membranes were probed with specific antibody for 15-PGDH and ß-actin (internal control). Autoradiographies of immunoblots revealed an increase of 15-PGDH protein level after 6 and 12 d of indomethacin treatment (I). Treatment cessation (I-) leads to a decrease of 15-PGDH expression (d 12) compared with treated cells. D, Expression of COX-1 in TT cells. It was determined in the cells cultured for 2 d in presence or not of 200 µM indomethacin. Western blots were obtained following the protocol described for 15-PGDH expression study. Indomethacin decreased significantly COX-1 protein expression (P < 0.01 vs. controls). The graph results were expressed as the ratio of COX-1/actin after autoradiography integration (Bio-1D software). Data are means ± SE (n = 6; **, P < 0.01). Immunoblots in panels C and D are representative from three independent experiments.

 


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  		FIG. 2. Decrease of DNA synthesis in TT cells elicited by indomethacin concentrations at 50, 100, and 200 µM. TT cells were seeded out at 75,000 cells/well (12-well plate). Tritiated thymidine (18.5 MBq) and indomethacin were added at the same time. Thymidine incorporation was measured after 6 h of treatment. Indomethacin decreased DNA synthesis activity in TT cells compared with control. Graph is representative of three independent experiments performed in triplicate. Results are the mean ± SE of three samples. ANOVA and Fisher tests were performed (ns, not significant; *, P < 0.05; **, P < 0.01 vs. control).

 
Expression and activity of 15-PGDH.
We investigated by Western blot analysis the effect of indomethacin and treatment cessation on 15-PGDH protein expression. A regular low increase of 15-PGDH activity was observed in the control group, probably due to a higher density of cells that modifies the cell phenotype. The 15-PGDH expression was enhanced by indomethacin after 6 and 12 d of treatment. Stopping the treatment at d 6 led to a strong decrease of 15-PGDH expression (d 12) compared with treated cells (Fig. 1CGo). The 15-PGDH activity increased by 40, 48, 31, and 26% with indomethacin at d 6, 8, 10, and 12, respectively (P < 0.005). When indomethacin was removed from the culture medium after 6 d, the activity of 15-PGDH was highly decreased to reach values of controls at d 10 and 12 (Fig. 1BGo). A negative correlation related 15-PGDH activity and TT cells proliferation (in percent of controls, n = 7; r = 0.80; P < 0.05).

Expression of COX-1 and COX-2 proteins in TT cells.
Western blot analysis indicated that TT cells expressed COX-1 protein. In our experimental conditions, COX-2 was not revealed in TT cells although it was well identified in renal tissue used as a positive control (data not shown). COX-1 protein expression (Fig. 1DGo) was significantly decreased by indomethacin treatment compared with the control cells (P < 0.01).

CT determination.
CT levels in culture medium were determined during TT cell proliferation at d 2, 4, and 6. Two hundred and 400 µM indomethacin produced a time-dependent decrease of cell number (Fig. 3AGo). A drastic decrease was observed with the highest dose. CT levels in culture medium were decreased as soon as d 2 with 400 µM indomethacin treatment. This effect was amplified until d 6 (Fig. 3BGo). The 200-µM indomethacin treatment significantly decreased CT levels at d 6. CT level was correlated with the TT cell number per well (n = 9; r = 0.77; P = 0.01).



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  		FIG. 3. Effect of indomethacin on CT levels in TT cells culture medium. Cells were seeded out at 150,000 cells/well, synchronized and treated or not treated with 200 or 400 µM of indomethacin for 2, 4, and 6 d. At each experimental time, culture medium was collected and frozen at –20 C until CT RIA. Cells were trypsinized, then counted. A, Indomethacin (200 µM) significantly decreased TT cells proliferation in a time-dependent manner. This effect is more important with the dose of 400 µM. B, CT levels were estimated by the specific RIA described in Materials and Methods. CT levels were decreased by indomethacin at d 6 with the lowest dose and from d 2–6 according to a time-dependent way with the highest dose. Results are the mean ± SE of three samples. ANOVA and Fisher tests were performed (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control).

 
Effect of dmPGE2 on TT cell cultures
PGE2 determination.
Intracellular PGE2 levels were diminished after 2 d of 200 µM of indomethacin treatment (15.8 ± 0.3 pg/105 cells vs. 150 ± 0.1 pg/105 cells in controls). In presence of 10 µM dmPGE2, we observed a diminution of PGE2 content (52.2 ± 12.2 pg/105 cells) that was more important when indomethacin was added (7.6 ± 1.3 pg/105 cells) (Fig. 4AGo).



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  		FIG. 4. Effect of dmPGE2 on TT cell proliferation and 15-PGDH activity. Cells were seeded out at 150,000 cells/well, synchronized, and treated or not treated with 200 µM indomethacin and with or without 10 µM dmPGE2 for 48 h. Cells were trypsinized, counted, and the proteins were extracted for 15-PGDH activity assay and PGE2 determination (enzyme immunoassay). A, PGE2 content (pg/105 cells) in TT cells treated or not treated with 200 µM indomethacin and/or 10 µM of dmPGE2. B, The addition of dmPGE2 diminished the number of cells vs. control. With both indomethacin and dmPGE2, a stronger decrease in cell proliferation is observed. C, dmPGE2 (10 µM) alone or with 200 µM indomethacin enhanced 15-PGDH activity compared with control at the same level as indomethacin alone. Results are the mean ± SE of three samples. ANOVA and Fisher tests were performed (*, P < 0.05; ***, P < 0.0001 vs. control).

 
Effect on cell proliferation and 15-PGDH activity.
A slight significant decrease of TT cell number was observed in presence of 10 µM dmPGE2 alone (Fig. 4BGo). Indomethacin alone (200 µM) or with 10 µM dmPGE2 induced the same decrease of proliferation. DmPGE2 and indomethacin alone or combined similarly increased the 15-PGDH activity (Fig. 4CGo).

Effect of indomethacin in vivo
Effect of indomethacin on tumor growth in nude mice.
The in vivo activity of indomethacin was next determined in nude mice. Indomethacin prevented tumor growth. Figure 5AGo illustrated the effect of indomethacin on the volumes of MTC xenografts in athymic mice. After oral administration of indomethacin (1.5 and 2 mg/kg body weight·d) during 7 wk, tumor volumes were reduced by 49 and 77%, respectively (P < 0.05 for the higher dose), and tumor weights were decreased by 30 and 77% (Fig. 5BGo). In a separate experiment, we showed that plasma CT level, the biological marker of MTC, and tumor weight in controls were very correlated (Fig. 5DGo, n = 15; r = 0.84; P < 0.0001). Seven weeks of indomethacin administration elicited a major decrease of plasma CT level, more important in magnitude than that of volumes and weights, respectively, 55 and 86% for the low and the high doses (Fig. 5CGo). Similar results were obtained when indomethacin was added to mice food (data not shown).



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  		FIG. 5. In vivo effect of indomethacin on growth of human medullary thyroid cancer xenografts in athymic mice. A, TT cells (5 x 106 cells/animal) were inoculated sc on the back of each nude mouse at d 0. Animals were divided into three groups: controls (n = 8) and indo- methacin-treated animals (1.5 mg/kg body weight/d, n = 8 and 2 mg/kg·d, n = 6). Indomethacin was administered in drinking water from d 1 for 7 wk. Indomethacin (2 mg/kg·d) significantly reduced the volumes of tumoral tissue after 5, 6, or 7 wk of treatment. Tumors were removed after 7 wk of treatment. Tumor weights (B) and plasma CT (C) were also decreased after 7 wk of indomethacin treatment. Tumor volumes, weights, and plasma CT are presented as mean ± SE (ns, not significant; *, P < 0.05 vs. control) (one-way ANOVA followed by Fisher test at each time). D, The relationship between plasma CT and tumor weight in controls. Circulating CT levels and tumor weights are significantly correlated (n = 15; r = 0.84; P < 0.0001). This result was obtained in a separate experiment that was representative of the others.

 
Histological and immunohistochemical findings of TT cell tumors.
Mann Dominici staining revealed that TT tumors were only composed of tumoral tissue frequently organized in lobules surrounded by a fibrous layer. A thin connective tissue surrounded the periphery of tumors. Very few vessels were observed. The treated tumors had the same appearance, and rare cavities were observed inside the treated tumors. The vascular network remained rare. Neutrophil or eosinophil were absent. Sometimes, infiltrating lymphocytes were present with the same frequency in control and treated tumors.

Antifactor VIII-related antigen was revealed by specific immunostaining in the rare microvessels, present in all the tumors studied. The estimation of tumor section area irrigated by capillaries (in percent of total slice surface) showed that its size was only 14 ± 5% in controls and 23 ± 4% in treated mice (Fig. 6AGo). Thus, indomethacin treatment did not significantly inhibit microvessel formation.



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  		FIG. 6. Analyses of induced TT cell tumors in nude mice treated or not treated with indomethacin. After 7 wk of treatment, tumors were removed from control (n = 8) and treated animals (2 mg/kg body weight·d; n = 6), frozen at –40 C then stored at –80 C. A, Angiogenesis study. After fluorescein isothiocyanate labeling of capillaries by antifactor VIII-related antigen antibody, the tumor section areas irrigated by microvessels were estimated. Indomethacin did not inhibit microvessel extension. B, Mitotic index (percentage) in TT tumors. The index of cell proliferation was determined from the ratio of nuclear Ki-67 protein-positive cells/total nuclei number visualized after 4',6-diamidino-2-phenylindole staining. A major decrease (74%) of the positive nuclei in the indomethacin-treated tumor tissues compared with controls is observed (*, P < 0.05). Micrographs of immunostaining of Ki-67 nuclear protein (fluorescein isothiocyanate fluorescence) revealed abundant dividing nuclei in control tumors although their number was decreased in indomethacin-treated tumors. C, Expression of 15-PGDH protein in TT tumors by Western blot analysis. Tumor section lysate protein (40 µg/lane) was loaded on a SDS-PAGE (4–20%), electroblotted, and transferred on a PVDF membrane. The membranes were probed with specific antibody for 15-PGDH and ß-actin (internal control). The graph results were expressed as the ratio of 15-PGDH/actin after autoradiography integration (Bio-1D software). The 15-PGDH expression was increased significantly in tumors treated with indomethacin compared with controls (*, P < 0.05). D, Expression of COX-1 protein in TT tumors by Western blot analysis. We investigated the COX-1 expression in TT tumors following the same protocol as described above. The graph results were expressed as the ratio of COX-1/actin after autoradiography integration (Bio-1D software). The COX-1 expression was decreased significantly in tumors treated with indomethacin compared with controls (*, P < 0.05). A, B, C, and D represent the mean of the results obtained for all the animals of each experimental group. C and D, Immunoblots above the graphs show three representative samples of each experimental animal group.

 
Only a few dead cells were revealed by the TUNEL method both in treated and control TT tumors. There was no difference between the treated and control groups.

The nuclei at the different phases of the cell cycle, except at G0, were revealed by immunostaining of the human Ki-67 protein (Fig. 6BGo). The mitotic index calculated from labeled cells was significantly decreased by 74% (P < 0.01) in TT cell tumors treated with indomethacin compared with controls (Fig. 6BGo).

A COX-2 immunostaining was visualized in cells such as infiltrating macrophages. Tumor tissue remained negative. Macrophages were identified in all tumors by anti-CD11b antibody. Treatment with indomethacin did not influence the expression of COX-2 immunoreactivity in these cells.

Immunohistochemistry showed the same positive staining for 15-PGDH and COX-1 proteins in the tumor tissue of treated and control mice; however, an effect of indomethacin could have been masked by the low sensitivity of the method (data not shown).

Expression of 15-PGDH and COX-1 proteins in tumors.
Levels of 15-PGDH expression were measured by Western blot analysis, in TT tumor tissue treated with or not treated with indomethacin. The 15-PGDH protein expression was significantly (P < 0.05) and strongly increased in treated tumors with indomethacin at the high dose of 2 mg/kg·d compared with untreated tumors (Fig. 6CGo). Western blot analysis of COX-1 protein in tumor tissues revealed a decrease in COX-1 expression after treatment by indomethacin, compared with controls (P = 0.05; Fig. 6DGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is in MTC (38) that, for the first time, an increase in PG levels was reported and associated with tumoral development. This increase was then observed in a number of cancers, and the antiproliferative effect of the NSAIDs was reported. However, to our knowledge, NSAIDs were never tested on MTC growth.

Our in vitro results demonstrated that indomethacin reduced proliferation of TT cells, the human MTC experimental model. After indomethacin treatment, the decrease in cell number was correlated with CT level in the culture medium. Indomethacin did not act or have a low action on the rate of CT secretion as the CT levels were a function of the number of tumoral cells. In vitro, in cells treated by indomethacin, CT remained a good marker of tumor cell proliferation. Indeed, in humans suffering from MTC, the determination of plasma CT levels are used for the diagnosis and the follow-up of the disease after surgical removal of the thyroid. Moreover, as previously reported (47), our work confirmed that TT cells transplanted into athymic nude mice produced solid sc tumor. We reported that CT was secreted in the blood by TT cell tumors at levels correlated to the tumor weight. Thus, the reduction of tumor volumes and weights associated with the decrease of circulating CT levels proved that oral administration of indomethacin prevented growth of TT cell xenografts. We revealed in tumors the expression of the PG synthesis enzyme, COX-1, and also that of the PG catabolism enzyme, 15-PGDH. COX-2 was only detected in the infiltrating macrophages and not in the tumoral tissue.

The mechanism of NSAID antiproliferative effect is controversial as they can act by inhibiting tumor cell division, by inducing apoptosis or by inhibiting angiogenesis. These effects are cell or tissue dependent and are bound or not to a decrease in PG levels due to the inhibition of COX enzyme activity.

Numerous studies attributed a prevention of carcinogenesis by inducing apoptosis (28, 31, 32). In TT cell xenotransplants, the number of TUNEL-positive nuclei was low in control and treated mice. Cell death, studied by TUNEL and a measure of caspase activity, was increased in treated cells but only in a medium containing 1% FCS. However, TUNEL staining performed in normal conditions (10% FCS) was not modified by the treatment. Our results are in agreement with a resistance to apoptosis. In MTC and TT cells, the deregulation of programmed cell death was attributed to a high expression of the Bcl-2 apoptosis regulatory gene by Wang et al. (48). Other authors suggested that proto-oncogene RET-mediated carcinogenesis in MTC critically depends on nuclear factor-{kappa}B activation, which inhibits apoptosis (49). Some in vitro studies found that NSAIDs inhibited cell division (28, 29, 30). Our data on TT cell xenotransplants showed a strong decrease of Ki-67 antigen proliferation index. The main action of indomethacin was a drastic inhibition of cell division as attested by the lower proliferation and DNA synthesis observed in TT cells and tumors.

Angiogenesis is a central determinant of solid tumor growth, and high PG levels promote angiogenesis. It has been suggested that although COX-2 regulates angiogenesis in colon cancer cells, COX-1 modulates angiogenesis in endothelial cells (35, 36). More recently, Nie and Honn (37) concluded that COX-1 and COX-2 in endothelial cells regulate angiogenesis. A weak increase of the vasculature was observed in TT cell xenografts, and indomethacin produced no reduction in microvessel density, although high levels of PGE2 are found in MTC (38) and in TT cells.

Numerous experiments have previously shown that PGE acts as a feedback inhibitor of the cellular immune response mediated by T lymphocytes and natural killer cells, in various conditions and particularly in tumor bearing animals. This response can be enhanced by the administration of NSAIDs (17). We have also investigated the immune effectors in TT tumor slices stained by Mann Dominici coloration. Few lymphocytes (natural killer cells in the tumors of nude mice) infiltrated TT cell xenografts. Their recruitment was similar in control- and indomethacin-treated tumors. This suggests that these effectors were not implicated in the antitumoral effect of this NSAID on MTC.

High levels of COX-2 mRNA and/or protein have been found in a variety of carcinoma (18, 19, 20, 21, 22, 23, 24, 25). Increased concentrations of COX-2 mRNA and protein were also found in human thyroid cancer (50). However, COX expression in thyroid C cells and MTC was not investigated. Moreover, infiltrating macrophages can favor carcinogenesis by paracrine COX-2 activity. In human colorectal adenomas, COX-2 is localized to the stromal cell compartment, predominently in macrophages (51, 52), and could mediate tumor promotion. Ko et al. (53) using in vitro models of macrophage-epithelial cell interactions, demonstrated that macrophage COX-2 has paracrine protumorigenic activity. In TT cell tumor model, COX-2 immunopositive infiltrating cells like macrophages have been identified, and these cells could be implicated in TT tumor growth. However, the similar immunostaining in control and treated tumors suggests that indomethacin did not inhibit or inhibited weakly COX-2 protein levels in macrophages. In fact, it was previously described that, in a number of cell lines, indomethacin blocks COX-2 active site without decreasing protein expression. So, we cannot exclude a decrease of COX-2 macrophage activity. Even, as previously reported (54), this NSAID is able to decrease COX-2 activity and, at the same time, increase protein expression.

Although expression of COX-2 protein is increased and COX-1 remains unchanged in most cancers, we observed COX-1 expression in TT cells and no detectable COX-2. In TT tumoral tissue, we also observed an elevated COX-1 and a reduced COX-2 expression. Such a result was obtained in human ovarian tumor where COX-1 protein is elevated and COX-2 protein remains at its basal level (55). Recently, the up-regulation of both COX-1 and COX-2 was reported in human prostate cancer (56), in a murine model of lung tumorigenesis (57), and in human cervical carcinoma (58). In TT cells, both in vitro and in vivo, COX-1 protein expression was decreased by indomethacin. To our knowledge, it is the first observation of such a phenomenon as indomethacin is known to inhibit COX-1 activity without changing COX-1 protein expression. This may be due to the fact that, in TT cells, COX-1 seems responsible for the PGE2 production. We performed RT-PCR amplification of mRNA extracted from TT cells. No change in COX-1 mRNA expression was observed after indomethacin treatment indicating a posttranscriptional effect (data not shown).

Although the major targets of NSAIDs are COXs, several reports indicate that NSAIDs may suppress tumor cell growth independently of COX inhibition (59). Baek et al. (60), who identified NSAID-activated gene 1 as a gene induced by some NSAIDs in colorectal cells devoid of COX activity, reported that indomethacin increased NSAID-activated gene 1 expression in a number of cells from tissues other than colorectal. The important and ubiquitous role of PG-catabolizing enzymes has been demonstrated by Taï et al. (61) and in particular the role of 15-PGDH. But only two publications (26, 27) described an implication of 15-PGDH in tumor growth. In the present study, 15-PGDH expression was increased when TT xenograft development was reduced.

The same relation was obtained between 15-PGDH expression and activity and proliferation of cultured TT cells. As generally described for COX inhibition, the increase of 15-PGDH expression and activity was reversible. Our results confirmed previous data showing a negative correlation between 15-PGDH activity and TT cell proliferation (42). Our results suggest the implication of 15-PGDH in the indomethacin antitumoral effect on TT cell tumors. In the present report, we showed that in vitro indomethacin decreased PGE2 levels. Treatment of TT cells with the stable analog of PGE2, dmPGE2, decreased both PGE2 content (three times) and cellular proliferation but enhanced 15-PGDH activity. On the contrary, in PC3 cells, as in other cells (DiFi and MDA-MB-134), dmPGE2 induced an increase of cell proliferation and endogenous PGE2. This effect is attributed to an up-regulation of COX-2 mRNA without effect on COX-1 mRNA (25). In TT cells not expressing COX-2 the decrease in endogenous PGE2 may be due to enhancement of 15-PGDH activity. We cannot exclude that exogenous dmPGE2 diminished cellular proliferation by a direct action on a differential PG receptor. But, it is more likely that in TT cells, the decrease of cell number observed was rather linked to the diminution in endogenous PGE2. When the cells were treated both with indomethacin and dmPGE2, an additional decrease in PGE2 cell content (20 times) and cell proliferation was obtained. No change in 15-PGDH activity was determined compared with dmPGE2 or indomethacin treatment alone. This result implicated that COX-1 enzyme activity was decreased by indomethacin and was responsible of the further diminution in PGE2 contents. The decrease of COX-1 protein expression after indomethacin treatment that we observed in vivo and in vitro reinforced this hypothesis.

In conclusion, our data demonstrated an antitumoral effect of indomethacin on TT tumor tissue, which does not implicate an immune response or angiogenesis. Furthermore, we showed that this NSAID acts on COX-1 and 15-PGDH expression in MTC. COX-1 inhibition and 15-PGDH activation could be of interest in the treatment of MTC. The therapeutic potential of long-term MTC treatment with NSAIDs should be considered to slow down tumor growth and delay the onset of metastasis. Thus, NSAIDs may represent an adjuvant therapy to surgery.


   Acknowledgments
 
We gratefully acknowledge Professor H. H. Taï (University of Kentucky, Lexington, KY) for his generous gift of 15-PGDH antibody and Professor A. Martin (Hôpital Avicenne, Bobigny, France) for histological analysis of COX-2. We thank also Dr M. S. Moukthar for his useful help in redacting this paper, and Professor G. Milhaud and Dr. P. Bobé for their helpful suggestions about the manuscript.


   Footnotes
 
This work was supported in part by the Grant 5355 of the Association de la Recherche sur le Cancer. V.Q. was supported by a grant from the Cancer and Solidarity Foundation and by Comité Départemental de la Ligue Contre le Cancer.

Abbreviations: CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; COX, cyclooxygenase; CT, calcitonin; DTT, dithiothreitol; FCS, fetal calf serum; hCT, human CT; 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; MTC, medullary thyroid cancer; NSAIDs, nonsteroidal antiinflammatory drugs; PG, prostaglandin; PVDF, polyvinylidene difluoride; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling.

Received July 21, 2003.

Accepted for publication January 12, 2004.


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Genetic Alterations in Thyroid Cancer: The Role of Mouse Models
Vet. Pathol., January 1, 2007; 44(1): 1 - 14.
[Abstract] [Full Text] [PDF]

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S. Swami, A. V. Krishnan, J. Moreno, R. B. Bhattacharyya, D. M. Peehl, and D. Feldman
Calcitriol and Genistein Actions to Inhibit the Prostaglandin Pathway: Potential Combination Therapy to Treat Prostate Cancer
J. Nutr., January 1, 2007; 137(1): 205S - 210S.
[Abstract] [Full Text] [PDF]

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D Wang and R N DuBois
PROSTAGLANDINS AND CANCER
Gut, January 1, 2006; 55(1): 115 - 122.
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