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Cyclooxygenase-2 Inhibitors Reverse Chemoresistance Phenotype in Medullary Thyroid Carcinoma by a Permeability Glycoprotein-Mediated Mechanism

Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy

Address all correspondence and requests for reprints to: Prof. Ettore C. degli Uberti, Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy. E-mail: ti8{at}unife.it.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Objective: Medullary thyroid carcinoma (MTC) is a highly chemoresistant malignant neoplasia deriving from parafollicular C cells. Chemotherapy failure has been ascribed, at least in part, to the overexpression by MTC of the multidrug resistance 1 (MDR1) gene, encoding a transmembrane glycoprotein [permeability glycoprotein (P-gp)] that antagonizes intracellular accumulation of cytotoxic agents. P-gp expression and function in a rat model have been demonstrated to depend on cyclooxygenase (COX)-2 isoform levels, which are found elevated in many human cancers. The aim of our study was to investigate the role of the COX-2 pathway in modulating chemoresistance.

Design and Results: We investigated P-gp and COX-2 expression and then evaluated the sensitizing effects of COX-2 inhibitors on the cytotoxic effects of doxorubicin in the presence or in the absence of prostaglandin E₂ in primary cultures and in a human MTC cell line, TT. Moreover, P-gp function has been studied. Our data show that TT cells express both MDR1 and COX-2 and that rofecoxib, a selective COX-2 inhibitor, sensitizes TT cells to the cytotoxic effects of doxorubicin, reducing P-gp expression and function.

Conclusions: Our data suggest that these effects are mediated by a mechanism not involving the generation of prostaglandin E₂, possibly implicating the synthesis of other COX-2 products.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
MEDICAL THERAPY OF many malignant tumors is frequently unsuccessful due to the chemoresistance phenotype characterizing these neoplasms. It has been demonstrated previously that multidrug resistance 1 (MDR1) gene, belonging to the ATP-binding cassette B1 family, is involved in multidrug resistance phenotype (1). Human MDR1 gene, located on chromosome 7 and consisting of 28 exons, encodes a 1280-amino acid protein, named permeability glycoprotein (P-gp). P-gp is a transmembrane glycosylated and phosphorylated 130- to 170-kDa protein that is expressed in liver, kidney, lung, gastrointestinal tract, gravid uterus, and blood–tissue barriers (blood–brain barrier, blood–testis barrier, and placenta) (2), as well as in many cancers (3). P-gp functions as a transmembrane efflux pump that translocates its substrates from the intracellular to the extracellular domain (4), therefore conferring resistance to many chemotherapeutic drugs in cancer cells. Cyclooxygenase (COX)-2 was found to regulate MDR1 expression in rat glomerular mesangial cells (5), suggesting that COX-2 might be involved in inducing the chemoresistance phenotype. We investigated whether COX-2 inhibition is able to reduce MDR1 expression and function in a medullary thyroid carcinoma (MTC) cell line and to sensitize MTC cells to the antiproliferative effects of doxorubicin.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
TT cell line

The TT cell line was obtained from the American Type Culture Collection (Manassas, VA) and maintained in culture as described previously (6). TT cells express and secrete calcitonin, carcinoembryonic antigen, chromogranin A, and many other peptides (7).

Primary culture

To explore the effects of COX-2 inhibitors on MTC primary culture, monolayer culture of tumor cells was performed from a portion of the fresh tissue as described previously (8, 9). Tissue samples were collected from patients undergoing total thyroidectomy for MTC in accordance with the guidelines of the local committee on human research. Briefly, tumor tissue was minced and enzymatically dissociated using 0.35% collagenase (Sigma, Milano, Italy) and 1% trypsin at 37 C for 60 min. Cell suspensions were filtered through double layers of gauze and washed twice with serum-free F-12 Ham’s Nutrient Modified Medium (F-12) (Euroclone Ltd., Wetherby, UK). Tumor cells were resuspended in F-12 with 10% fetal bovine serum and antibiotics, seeded in 96-well culture plates (2 x 10⁴ cells per well), and incubated at 37 C in a humidified atmosphere of 5% CO₂ and 95% air. After 24 h, cells were incubated overnight with serum-free F-12 medium. The day after, cells were treated with test substances, and cell viability was assessed.

Informed consent of the patients was obtained for disclosing clinical investigation and performing the in vitro study.

Primary cultured cells were employed for only in vitro cell viability experiments because a limited number of cells were available. Functional experiments and protein expression assessment were, therefore, performed only in the TT cell line.

Compounds

R(+)-Verapamil hydrochloride and rhodamine 123 (R123) were purchased from Sigma (Milano, Italy). Doxorubicin hydrochloride (Adriblastin) was obtained from Pharmacia (Milano, Italy). Prostaglandin E₂ (PGE₂) was purchased from MP Biomedicals Europe (Relegem, Belgium). Rofecoxib was kindly provided by Merck Sharp & Dohme (Roma, Italy). N-[2-(cyclohexyloxy)4-nitrophenyl]methanesulfonamide (NS-398), a specific COX-2 inhibitor, was purchased from Alexis Biochemicals (Lausen, Switzerland).

Isolation of RNA and RT-PCR

To evaluate MDR1 and COX-2 expression in TT cells, RT-PCR analysis was performed. Total RNA was isolated from subconfluent TT cells and from pulverized tissues by using TRIzol reagent (Invitrogen, Milano, Italy), according to the protocol of the manufacturer, and subjected to RT with random hexamers, as described previously (10, 11). To prevent DNA contamination, RNA was treated with ribonuclease-free deoxyribonuclease (Promega, Milano, Italy). The cDNA was then amplified by PCR using the PCR Master Mix (Promega, Milano, Italy) in the conditions recommended by the suppliers. PCRs were carried out using the oligonucleotide primers and conditions listed in Table 1, which describes the size of expected fragments. PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining. Each PCR product was subjected to restriction enzyme digestion and analyzed on 2% agarose gel to further confirm correct identification of the amplicons (data not shown).

To evaluate the effects of COX-2 inhibitors on MDR1 expression in TT cells, relative quantitative PCR and the Comparative C_T Method (User Bulletin No. 2, Applied Biosystems, Monza, Italy) in multiplex format with the Pre-Developed TaqMan Assay Reagent no. 4319413E was performed using Assay on Demand Hs00184491_m1 (Applied Biosystems). All relative quantitative PCRs were performed, recorded, and analyzed using the ABI 7700 Prism Sequence Detection System (Applied Biosystems). All samples were carried out in triplicate (10 ng of RT total RNA per well) and repeated at least twice. Controls without template or RT were run in each experiment.

Viable cell number assessment

Variations in cell number were assessed by the Cell Proliferation Kit (Roche, Germany), as described previously (12). TT cells or primary cultured cells were incubated with or without the test substances for 1–7 d. After incubation, the revealing solution was added, and the absorbance at 560 nm was recorded using the Wallac Victor 1420 Multilabel Counter (Perkin-Elmer, Monza, Italy) in at least three experiments in six replicates.

Western blot analysis

For immunoblotting, TT cells were resuspended in sample buffer [60 mM Tris (pH 6.8) containing 5% sodium dodecyl sulfate, 10% glycerol, 2.5% ß-mercaptoethanol, and 0.02% bromphenol blue]. Samples were lysed at 100 C for 10 min, and protein concentration was measured by bicinchoninic acid Protein Assay Reagent Kit (Pierce, Rockford, IL), as described previously (13). Cell proteins were then fractionated on 7% SDS-PAGE, as described previously (14), and transferred by electrophoresis to a Nitrocellulose Transfer Membrane (PROTRAN, Dassel, Germany). Membranes were incubated with anti-P-gp monoclonal antibody (Chemicon International, Inc., Temecula, CA) at 1:800. Horseradish peroxidase-conjugated antibody IgG (Sigma) was used at 1:2000, and binding was revealed using enhanced chemiluminescence (Amersham Biosciences, Uppsala, Sweden). The blots were then stripped and used for further blotting with antiactin antibody (Sigma). Quantitative densitometry of the x-ray films from the luminograms was performed using the Quantity-One software (Bio-Rad, Hercules, CA) on the Fluor-S Multiimager Bio-Rad scanning densitometer.

R123 assay

TT cells were seeded in 24-well plates at 2 x 10⁵ cells per well in culture medium the day before the experiment. Cells were washed twice with serum-free culture medium and then incubated with or without 4 µM R123 for 1 h at 37 C, as described previously (15). After incubation, the culture medium was aspirated gently, and cells were washed three times with 1 ml of ice-cold serum-free F-12 to remove any extracellular R123. Cells were then incubated with serum-free F-12 with or without 100 µM verapamil at 37 C. After 1 h, culture medium was removed, and cells were washed three times with 1 ml ice-cold PBS, solubilized with 1 ml 0.2 M NaOH overnight, and assayed for R123 and protein contents as described above. The concentration of R123 in each sample was determined quantitatively by fluorescence spectrophotometry using the Wallac Victor 1420 Multilabel Counter (_ex = 485 nm, _em = 535 nm) and standardized by the protein content of each sample. All experiments were carried out in triplicate.

Statistical analysis

Results are expressed as the mean ± SE. A preliminary analysis was carried out to determine whether the datasets conformed to a normal distribution, and a computation of homogeneity of variance was performed using Bartlett’s test. The results were compared within each group and between groups using ANOVA. If the F values were significant (P < 0.05), Student’s paired or unpaired t test was used to evaluate individual differences between means, and P < 0.05 was considered significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
MDR1 and COX-2 expression in TT cells

To assess MDR1 and COX-2 expression in MTC and to verify whether TT cells are a suitable model for evaluating the effects of COX-2 inhibitors on MDR1, we evaluated the expression of these two genes in TT cells and in MTC samples by RT-PCR. As shown in Fig. 1, all selected MTC samples and the TT cell line expressed both MDR1 and COX-2.

View larger version (45K): [in this window] [in a new window]   FIG. 1. MDR1 and COX-2 expression in TT cells and in MTC samples. Isolated RNA (1 µg/reaction) from 12 MTC samples and from TT cells was treated with deoxyribonuclease and subjected to RT. Aliquots from the generated cDNA were subjected to subsequent PCR amplification of MDR1 and COX-2 using the primers indicated in Table 1. PCR products were resolved on a 2% agarose gel. The expected PCR products are indicated with arrows. M, 100-bp PCR marker; lanes 1–12, MTC samples; TT, TT cell line.

To evaluate whether TT cells are resistant to treatment with common chemotherapeutic agents, cells were treated with doxorubicin at concentrations ranging from 1–2000 nM up to 7 d. As shown in Fig. 2, TT cells were resistant to treatment with doxorubicin up to 300 nM, whereas higher concentrations reduced TT cell growth (P < 0.05 and P < 0.01).

View larger version (18K): [in this window] [in a new window]   FIG. 2. Effects of doxorubicin on TT cell proliferation. TT cells were incubated in 96-well plates for 7 d in culture medium supplemented with doxorubicin at concentrations ranging from 1–2000 nM. Data from three individual experiments evaluated independently with six replicates are expressed as the mean ± SE percentage of cell proliferation inhibition vs. untreated control cells. *, P < 0.05; **, P < 0.01 vs. control.

Preliminary experiments performed by incubating TT cells up to 7 d with increasing concentrations (10^–3 to 200 µM) of verapamil, a specific P-gp inhibitor, demonstrated that this compound does not influence TT cell viability at concentrations of 100 µM or less (Fig. 3A). A concentration of 100 µM was, therefore, selected to inhibit P-gp function in cell viability experiments.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Effects of verapamil, rofecoxib, NS-398, and PGE2 on TT cell proliferation. TT cells were incubated in 96-well plates for 7 d in culture medium supplemented with or without verapamil at concentrations ranging from 10–3 to 200 µM (A), rofecoxib at concentrations ranging from 10–5 to 250 µM (B), NS-398 at concentrations ranging from 10–3 to 500 µM (C), or PGE2 at concentrations ranging from 10–6 to 10 µM (D). Data from three individual experiments evaluated independently with six replicates are expressed as the mean ± SE percentage of cell proliferation inhibition vs. untreated control cells. **, P < 0.01 vs. control.

To evaluate whether COX-2 inhibitors might sensitize TT cells to the cytotoxic effects of doxorubicin, TT cells were incubated with or without 50 nM doxorubicin, 100 µM verapamil, and 25 µM rofecoxib for 7 d. As shown in Fig. 4A, doxorubicin alone did not influence TT viable cell number. However, a significant reduction in cell proliferation was observed after cotreatment with verapamil (–27.3%; P < 0.05 vs. control) or with rofecoxib (–31.4%; P < 0.05 vs. control).

View larger version (22K): [in this window] [in a new window]   FIG. 4. Effects of combined treatment with doxorubicin and verapamil or rofecoxib on TT cell proliferation. A, TT cells were incubated in 96-well plates for 7 d in culture medium supplemented with or without 50 nM doxorubicin, 100 µM verapamil, 25 µM rofecoxib alone or in combination. B, TT cells were incubated in 96-well plates for 7 d in culture medium supplemented with or without 50 nM doxorubicin and/or 10 µM NS-398 alone or in combination. Data from three individual experiments evaluated independently with six replicates are expressed as the mean ± SE percentage of cell proliferation inhibition vs. untreated control cells. *, P < 0.05 vs. control.

Effects of rofecoxib on MTC cell viability

To evaluate whether COX-2 inhibitors might sensitize MTC cells to the cytotoxic effects of doxorubicin, five MTC primary cultures were incubated with or without 50 nM doxorubicin, 100 µM verapamil, and 25 µM rofecoxib for 3 d. As shown in Fig. 5, doxorubicin, verapamil, and rofecoxib did not influence MTC viable cell number. However, a significant reduction in cell viability was observed after cotreatment with doxorubicin and verapamil (–20%; P < 0.05 vs. control) or with doxorubicin and rofecoxib (–22%; P < 0.05 vs. control).

View larger version (17K): [in this window] [in a new window]   FIG. 5. Effects of doxorubicin and rofecoxib on MTC cell viability. MTC primary cultures were incubated in 96-well plates for 7 d in culture medium supplemented with or without 50 nM doxorubicin, 100 µM verapamil, and/or 25 µM rofecoxib alone or in combination. Data from five individual experiments evaluated independently with six replicates are expressed as the mean ± SE percentage of cell proliferation inhibition vs. untreated control cells. *, P < 0.05 vs. control.

Preliminary experiments performed by incubating TT cells up to 7 d with increasing concentrations (10^–6 to 10 µM) of PGE₂, a COX-2 end product, demonstrated that this compound does not influence TT cell viability at any concentration tested (Fig. 3D). To evaluate whether PGE₂ could influence the effects of rofecoxib, TT cells were incubated with or without 50 nM doxorubicin, 25 µM rofecoxib, and/or 1 µM PGE₂ for 7 d. As shown in Fig. 6A, PGE₂ alone or in combination with rofecoxib did not influence TT viable cell number. However, a significant reduction in cell proliferation was observed after cotreatment with PGE₂ and doxorubicin (–25.8%; P < 0.01 vs. control) or with PGE₂ and doxorubicin plus rofecoxib (–38.5%; P < 0.01 vs. control). Moreover, cotreatment with PGE₂, doxorubicin, and NS-398 for 7 d determined an even greater reduction in TT cell viability (–75%; P < 0.01 vs. control) (Fig. 6B).

View larger version (21K): [in this window] [in a new window]   FIG. 6. Effects of PGE2 on TT cell proliferation. A, TT cells were incubated in 96-well plates for 7 d in culture medium supplemented with or without 1 µM PGE2, 50 nM doxorubicin, or 25 µM rofecoxib alone or in combination. B, TT cells were incubated in 96-well plates for 7 d in culture medium supplemented with or without 1 µM PGE2, 50 nM doxorubicin, or 10 µM NS-398 alone or in combination. Data from three individual experiments evaluated independently with six replicates are expressed as the mean ± SE percentage of cell proliferation inhibition vs. untreated control cells. **, P < 0.01 vs. control.

To verify that P-gp is functional in TT cells, R123 assay was performed on TT cells treated with or without 100 µM verapamil for 1 h. As shown in Fig. 7A, verapamil significantly induced R123 intracellular retention (46%; P < 0.05). To assess the effects of COX-2 inhibitors on P-gp function, R123 assay was performed on TT cells treated with or without 25 µM rofecoxib for 7 d. As shown in Fig. 7B, rofecoxib markedly and significantly induced R123 intracellular retention (70%; P < 0.01).

View larger version (12K): [in this window] [in a new window]   FIG. 7. R123 assay. A, TT cells were incubated with or without 4 µM R123, washed, incubated for 1 h with or without 100 µM verapamil, and then lysed. Sample fluorescence was then measured and standardized by the protein content. B, TT cells were incubated with or without 25 µM rofecoxib for 7 d and then seeded in 24-well plates. The day after, cells were incubated with or without 4 µM R123, washed, and then lysed. Sample fluorescence was then measured and standardized by the protein content. Data from three individual experiments evaluated independently with three replicates are expressed as the mean ± SE percentage of cell proliferation inhibition vs. untreated control cells. 0, Untreated control cells; V, cells treated with 100 µM verapamil; R, cells treated with 25 µM rofecoxib. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

To verify whether the expression of MDR1 could be influenced by COX-2 inhibitors, relative quantitative PCR was performed on cDNA from TT cells treated previously with or without rofecoxib and/or PGE₂. As reported in Fig. 8, a reduction in MDR1 expression was evident in rofecoxib-treated cells. Moreover, PGE₂ treatment induced a similar inhibitory effect, further reducing MDR1 mRNA levels when combined with rofecoxib.

View larger version (10K): [in this window] [in a new window]   FIG. 8. Effects of rofecoxib and PGE2 on MDR1 expression. Real-time PCR analysis of MDR1 expression in TT cells incubated for 7 d with or without 25 µM rofecoxib (R), 1 µM PGE2, or both. Data from three individual experiments evaluated independently with three replicates are expressed as the mean ± SE percentage of MDR1 expression vs. untreated control cells. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

To assess the effects of COX-2 inhibitors on P-gp levels, Western blot analysis for P-gp was performed with protein extracts from TT cells treated with or without 25 µM rofecoxib for 7 d. As indicated in Fig. 9, treatment with rofecoxib markedly reduced P-gp levels.

View larger version (50K): [in this window] [in a new window]   FIG. 9. Effects of rofecoxib on P-gp levels. Western blot analysis for P-gp and actin of protein extracts from TT cells incubated for 7 d with or without 25 µM rofecoxib (R).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

Our data show that selective COX-2 inhibitors are capable of sensitizing chemoresistant MTC cells to the cytotoxic effects of a widely employed chemotherapeutic drug, such as doxorubicin. Previous studies showed that in MTC patients, treatment with doxorubicin induces only partial and transient response (26), indicating that MTC is resistant to treatment with this drug. The efficacy of medical therapy for MTC, the management of which is still controversial (27), can be increased by cotreating with doxorubicin and other potent chemotherapeutic drugs, with a consequent increase of serious side effects (28). The evidence presented here suggests that COX-2 inhibitors might be useful to sensitize neoplastic cells to doxorubicin. Indeed, our experimental model, the TT cell line, which expresses both COX-2 and MDR1, displays resistance to the cytotoxic effects of doxorubicin. Treatment with verapamil, a well-known P-gp inhibitor (29), induces R123 retention, indicating that verapamil is indeed capable of reducing P-gp function in TT cells. Moreover, verapamil is capable of sensitizing TT cells and MTC primary cultures to the cytotoxic effects of doxorubicin, suggesting that inhibition of P-gp function and/or MDR1 expression influences TT cell sensitivity to chemotherapeutic drugs. In addition, our data show that the selective COX-2 inhibitor rofecoxib is capable of inhibiting P-gp function because it induces R123 retention into TT cells and sensitizes both TT cells and MTC primary cultures to the cytotoxic effects of doxorubicin. Previous evidence showed that rofecoxib per se induces a dose-dependent increase in apoptosis and growth inhibition in the Lewis lung tumor cell line in vitro (30). However, in our model, no cytotoxic effect of this compound was observed. Furthermore, the selective COX-2 inhibitor reduces both MDR1 expression and P-gp levels, indicating that rofecoxib modulates MDR1 expression. These data are consistent with the previously shown evidence of a causal link between COX-2 and P-gp activity (5).

The reduced P-gp levels might account for the decreased P-gp function in rofecoxib-treated TT cells, which, therefore, become sensitive to doxorubicin. Indeed, due to the decreased transmembrane efflux pump function, we can hypothesize that the chemotherapeutic agent accumulates in the cell and exerts its antiproliferative effects.

To clarify whether these effects are due to a reduction in COX-2 end products induced by rofecoxib, we assessed the effects of PGE₂. Our results show that this prostaglandin does not influence TT cell viability per se and does not revert the doxorubicin-sensitizing effects of rofecoxib. On the contrary, PGE₂ seems to sensitize TT cells to doxorubicin. Moreover, PGE₂ does not revert the inhibitory effects of rofecoxib on MDR1 expression but causes a further reduction of MDR1 mRNA levels. These results differ from data indicating an induction of functionally active P-gp in primary rat hepatocytes and in HL-60 cells by PGE₂ treatment (31, 32). In these studies, however, no evidence for an induction in MDR1 expression has been provided. Moreover, the experimental settings and cell systems are completely different from ours.

Our findings suggest that rofecoxib exerts its effects through a mechanism not involving the generation of PGE₂, possibly implicating the synthesis of other COX-2 products, such as PGI₂ (33). This hypothesis is further strengthened by the evidence that the sensitizing effects toward the cytotoxic effects of doxorubicin exerted by another specific COX-2 inhibitor, NS-398, are not reverted by PGE₂. Moreover, NS-398 has been reported to block the COX-2-mediated increase in MDR1 expression and activity, suggesting that COX-2 products may be implicated in this response (5). However, our results do not rule out the possibility that rofecoxib might act directly and independently of COX-2 inhibition and prostaglandin generation, as suggested previously (21). Additional studies are needed to explore this possibility.

Our results suggest that selective COX-2 inhibitors might be useful in combination with chemotherapy and/or as neoadjuvant therapy in the medical treatment of metastatic MTC when surgery is not feasible. Clinical studies are needed to assess safety, toxicity, and efficacy of COX-2 inhibitors in these settings, especially after the clinical trial on adenomatous polyp prevention by the use of rofecoxib showed an increased cardiovascular risk (34). This issue is of great importance because treatment with doxorubicin has been linked to severe cardiac injury. Indeed, in animal models, cardiomyocyte injury is limited by the induction of COX-2 by doxorubicin and aggravated by coadministration of a COX-2 inhibitor. Injury was attenuated by prior administration of a prostacyclin analog (35). However, concentrations of COX-2 inhibitors needed for MDR1 down-regulation seem to be rather low when compared with antiinflammatory doses. Moreover, COX-2 inhibitors would be coadministered with chemotherapeutic drugs in cyclic fashion and for a limited period of time after the use of standard chemotherapeutic regimens (21). Increased risk for serious cardiovascular adverse events appears after 18 months of chronic treatment with COX-2 inhibitors, and, therefore, it is unlikely to occur in these settings (27). Moreover, our data show that PGE₂ further reduces MDR1 expression and sensitizes TT cells to doxorubicin, potentiating the effects of rofecoxib. Therefore, treatment with doxorubicin, COX-2 inhibitors, and PGE₂ in combination might achieve better results in terms of suppression of P-gp function, preventing the cardiovascular side effects due to a reduced prostaglandin production.

In conclusion, our findings provide evidence that a selective COX-2 inhibitor reduces MDR1 expression, P-gp level, and function in MTC cells by a mechanism not involving PGE₂. Our data point to a possible application of selective COX-2 inhibitors in combination with chemotherapy and/or as neoadjuvant therapy in the medical treatment of metastatic MTC, as suggested previously for differentiated thyroid carcinoma (36, 37).

	Footnotes

 
This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research (University of Ferrara: 60%–2004, and Ministero dell’Istruzione, dell’Università e della Ricerca Grant 2002067251-003), Fondazione Cassa di Risparmio di Ferrara, and Associazione Ferrarese dell’Ipertensione Artensione Arteriosa.

First Published Online August 9, 2005

Abbreviations: COX, Cyclooxygenase; F-12, F-12 Ham’s Nutrient Modified Medium; MDR1, multidrug resistance 1; MTC, medullary thyroid carcinoma; NS-398, N-[2-(cyclohexyloxy)4-nitrophenyl]methanesulfonamide; PGE₂, prostaglandin E₂; P-gp, permeability glycoprotein; R123, rhodamine 123.

Received June 17, 2005.

Accepted July 11, 2005.

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