ANTICANCER RESEARCH 29: 3095-3102 (2009)

Co-inhibition of Cyclooxygenase-2 and Dihydropyrimidine Dehydrogenase by Non-steroidal Anti-inflammatory Drugs in Tumor Cells and Xenografts

ANDREA RÉTI1, ÉVA PAP1, ATTILA ZALATNAI2, ANDRÁS JENEY2, JUDIT KRALOVÁNSZKY1 and BARNA BUDAI1

1National Institute of Oncology, Budapest; 2First Institute of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary

Abstract. Background: Non-steroidal anti-inflammatory with S-1 (2). Recently there has been much drugs (NSAIDs) may be able to enhance the antitumor effect interest in using COX-2 inhibitors along with conventional of cancer drugs. Cyclooxygenase-2 (COX-2) is the best anticancer therapy based on the idea that many of the characterized target of NSAIDs. It was demonstrated that COX-2-regulated that contribute to tumor elevated dihydropyrimidine dehydrogenase (DPD) and COX- progression may also be determinants of tumor chemo- or 2 activities influence the response to 5- (5-FU). radiosensitivity (3). We previously showed that NSAIDs increased 5-FU sensitivity Several preclinical and clinical studies have shown that only in high COX-2-expressing cancer cells. Materials and non-steroidal anti-inflammatory drugs (NSAIDs) are Methods: The effect of indomethacin and NS-398 on DPD effective chemopreventive and antitumor agents, either alone activity and mRNA expression in a high COX-2-expressing or in combination with standard cancer therapies (4-6). The (determined by Western blotting, immunoflourescence and cyclooxygenase are the best characterized targets immunohistochemistry) cell line (24-, 48-hour, 10-day of NSAIDs, which inhibit their activity. treatment) and xenograft (3-week treatment) was investigated. 5-Fluorouracil (5-FU) is one of the most widely used Results: The coexistence of high COX-2 and DPD activity or anticancer agents for the treatment of colorectal cancer. low activities of both enzymes were detected. After treatment Dihydropyrimidine dehydrogenase (DPD) is the first and with NSAIDs, a simultaneous and significant decrease of both rate-limiting in the catabolic pathway of 5-FU. activities was also demonstrated. Conclusion: NSAIDs could Determination of tumoral DPD has become of clinical be promising modulators of fluorouracil-based chemotherapy, interest because elevated intratumoral DPD can decrease the especially in high COX-2-expressing tumours. tumor response to 5-FU therapy as a result of increased drug inactivation. The degradation of 5-FU through this pathway Cyclooxygenase-2 (COX-2) overexpression is common in reduces its efficiency and requires the application of a variety of human malignancies, including cancer of the extremely high doses. Furthermore, the breakdown of 5-FU colon, and promotes tumor cell growth, angiogenesis, leading to fluorinated catabolites might cause side-effects in tumor invasion and metastasis. The unfavorable prognostic the nervous system (7, 8). significance of elevated COX-2 expression was proven by In order to improve the effectiveness of 5-FU therapy, a Achiwa et al. showing an association between high COX- new subclass of orally administrated fluoropyrimidines, UFT 2 mRNA expression levels and shorter survival of patients (9) and S1 (10) containing uracil and 5-chloro-2,4 with curatively resected non-small cell lung cancer (1). dihydroxypyridine, respectively, as potent inhibitors of DPD, Similarly Uchida et al. found significant association and a direct mechanism-based irreversible DPD inhibitor, between the high COX-2 expression in colorectal tumors ethynyluracil, were developed (11). Kralovánszky et al. also and the less favourable clinical outcome of patients treated showed that 5-ethyl-2’-deoxyuridine enhanced the therapeutic index of 5-FU by reducing its catabolism (12). Several studies showed that NSAIDs can increase the efficacy of chemotherapeutic drugs. Specifically, combining Correspondence to: Judit Kralovánszky, Ph.D., National Institute of flurbiprofen, sulindac or indomethacin (INDO) with Oncology, Rath Gy. str. 7-9, Budapest, H-1122, Hungary. Tel: +36 12248787, Fax: +36 12248620, e-mail: [email protected] methothrexate, cyclophosphamide, melphalan, vincristine, and 5-FU resulted in an enhancement of Key Words: NSAIDs, COX-2, DPD, HCA-7, HT-29. cytotoxicity (5, 6, 13, 14).

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Table I. Summary of COX-2 and DPD status of the investigated cell lines and xenografts based on our previous* (15) and present study.

COX-2 expression

WB IHC IF PGE2 level DPD activity

Cells HCA-7 H* - H* H H HT-29 L* - L* L L Xenografts HCA-7 L - - - L HT-29 H H - H H

WB, Western blot; IHC, immunohistochemistry; IF, immunofluorescent staining; H, L, high and low expression/activity.

Previously we have shown that COX inhibitors INDO and NS-398 applied in low concentrations effectively increased the 5-FU-sensitivity only of HCA-7 colon cancer cells expressing a high level of COX-2 but not of the HT-29 cell line, which fails to express a high level of COX-2 protein (15). In order to understand the enhanced cytotoxic effect of 5-FU plus Figure 1. Western blot analysis of COX-2 and β-actin expressions of cell NSAIDs, the aim of the present study was to investigate in lines and xenografts. HCA-7 cells were treated for 10 days and HT-29 CRC cell lines and xenografts if, besides their PGE2-reducing xenografts for 3 weeks with indomethacin (INDO) or NS-398. a.u.: effect, NSAIDS may also modify DPD enzyme activity, the Arbitrary unit representing the optical density ratio of COX-2/β-actin. most important determinant of 5-FU action.

Materials and Methods Pathology and Experimental Cancer Research, Semmelweis University, Budapest. Xenograft-bearing mice were randomly separated into 3 Cell lines and treatments. HCA-7 colony 29 and HT-29 cells were groups with 3 mice each: vehicle control, and INDO- and NS-398- purchased from the European Collection of Cell Cultures (Salisbury, treated. The dose of INDO and NS-398 was 2.5 and 5 mg/kg body UK). HCA-7 and HT-29 cells were cultured in Dulbecco’s modified weight, respectively, given in 0.2 ml saline solution. These doses could Eagle’s medium or RPMI, respectively, supplemented with 10% be regarded as relatively low doses, based on earlier published studies of (v/v) fetal bovine serum. Eli et al. (16) and Sawaoka et al. (17). The treatments were given by All culture and chemical reagents were purchased from Sigma- oral gavage for 5 days/week for 3 consecutive weeks. Aldrich (St. Louis, MO, USA), unless otherwise indicated. NS- At the end of the treatments, mice were killed by cervical 398 was purchased from Cayman Chemicals (Ann Arbor, MI, dislocation and the tumors were rapidly removed. The tissue USA). Similarly to our previous study (15), relatively low samples were quick frozen by immersion in liquid nitrogen and concentrations of INDO and NS-398, namely 10 and 1.77 μM, stored until processing. The tumor tissues were pulverized with respectively were applied. Mikro-Dismembrator S (Braun Biotech International, Melsungen Cells were seeded at 1×106/well in 6-well plates and grown for 24 Germany) for 60 s and suspended in Hanks buffer. After h. The media were replaced and cells were treated with the drugs for centrifugation at 100,000×g for 30 min, the cytosol was removed 24 or 48 hours. In order to simulate the long term effect of NSAID and used for further investigations of PGE2 level and DPD activity. treatment on xenografts 3×105 HCA-7 cells were plated into 6-well plates and grown for 24 h, then the media were replaced and the cells Determination of COX-2 expression and activity. COX-2 protein were treated with the drugs for 10 days. At the end of the treatments, expression of cells was investigated by Western blotting using cell culture media were used to determine the concentration of specific anti-human COX-2 . In the case of prostaglandin E2 (PGE2). Cells were lysed by 3× freeze-thawing in xenografts, tumor tissues were pulverized as described above and Hanks buffer and centrifuged at 13,000×g for 30 min at 4˚C. The suspended in ice-cold lysis buffer [10 mM Tris pH 8.0, 0.15 M cytosol was collected in separate vials to determine the DPD activity. NaCl, 1% NP-40, 1 mM EDTA + 0.1% SDS + 50 mM Na-diethyl- dithiocarbamate + protease inhibitor mix (Pierce, Rockford, IL, Xenografts and drug administration. A suspension of 2×106 HCA-7 or USA)]. Samples were centrifuged for 10 min at 10,000×g at 4˚C HT-29 cells in physiological saline (0.1 ml) was injected subcutaneously and the supernatant was used for the further studies. The Western into the hind leg of 6-week-old female SCID mice obtained from the blot and the COX-2 immunofluorescent detection were performed in-house breeding of the Animal Care Facility of the First Institute of as described elsewhere (15).

3096 Réti et al: Co-inhibition of COX-2 and DPD by NSAIDs

Figure 2. A, Immunofluorescent staining of COX-2 protein on HCA-7 cells after 48-hour treatment with indomethacin (INDO) or NS-398. B, Immunohistochemistry for COX-2 expression in HT-29 xenografts after 3-week treatment with indomethacin (INDO) or NS-398.

Immunohistochemistry was also applied for the demonstration of Supermix (Bio-Rad, Hercules, CA, USA) and 3.6 μl H2O. The 3 COX-2 protein. Formalin-fixed, paraffin-embedded specimens were min denaturation at 95˚C was followed by 50 cycles of 20 s incubated with polyclonal anti-human COX-2 antibody (Lab Vision, denaturation at 95˚C, 20 s annealing at 62˚C and 30 s extension at Warm Springs CA, USA). Biotinylated antirabbit IgG and 72˚C. The quantitative DPD mRNA values were calculated relative streptavidin-horseradish peroxidase complex were used as to the GAPDH expressions. The analysis was performed three times secondary antibodies (DAKO, Denmark). Antigen visualisation was and the mean values were calculated. achieved by diaminobenzidine chromogen (DAB; Sigma) in 0.1% The DPD activity of the cytosols prepared from the cells and H2O2 PBS solution. The specimens were counterstained with xenografts were investigated by radioenzymatic method described hematoxylin. The specificity of the applied antibodies was checked elsewhere (15). Measurements were made in parallel in three with positive or negative controls. Specimens treated without independent experiments. In order to avoid bias caused by the further primary antibodies served as negative controls. of dihydrofluorouracil, the DPD activity was also calculated 14 The PGE2 concentrations were considered as a measure of COX-2 from the changes of [6- C] 5-FU concentration. Minor non-significant activity because of the unchanged COX-1 expressions. The PGE2 differences were found between the two calculation methods. levels were measured by ELISA (Assay Designs, Ann Arbor, MI, USA) from cell culture media and from the cytosols of the xenografts Measurement of endogenous uracil concentration. After 24-h according to the protocol of the manufacturer. Measurements were NSAID treatment of HCA-7 cells, endogenous uracil levels were made in duplicate in three separate experiments. measured. Cells were harvested by trypsinization and centrifuged for 5 min at 10,000×g at 4˚C. Ethylacetate in 2 M Tris buffer was Determination of DPD expression and activity. DPD mRNA added and the mixture was vortexed and centrifuged for 15 min at expression was determined by real time RT-PCR. After 24-hour 3,000×g. The ethylacetate layer was evaporated under a nitrogen treatment of HCA-7 cells with NSAIDs, total RNA was extracted stream. The residue was dissolved in eluent (0.4% methanol and and reverse-transcribed to cDNA using Superscript III (Invitrogen 0.04% tetrahydrofuran in 12.5 mM phosphate buffer) and the Inc, Carlsbad, CA, USA) according the manufacturer’s instructions. mixture was then separated by HPLC (Merck-Hitachi, Tokyo, The primers for GAPDH mRNA as a reference were provided in the Japan) on an Aquasil C18 column (150×4.6 mm 3 μm Thermo kit. The primers used for amplification of DPD were: forward 5’- Hypersil-Keystone, Runcorn, UK). Uracil was monitored by UV GAA ATG GCC GGA TTG AAG TTT and reverse 5’-TCG AAG detector at 214 nm, eluted after 8 min at a flow rate of 0.6 ml/min. AGC TTT TGA AGC TGG. Amplifications were performed in a Bromouracil was used as an internal standard. Measurements were mixture of 1 μl cDNA, 0.2 μl of primers, 5 μl IQ SybrGreen made in three independent experiments.

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Table II. Effect of INDO and NS-398 treatment on PGE2 production and PGE2 level and DPD activity. In HCA-7 cells, INDO and DPD activity on high COX-2 expressing HCA-7 cells and HT-29 NS-398 inhibited the PGE2 production in a time-dependent xenografts. manner: 24-hour treatment resulted in more than an 80% Treatment inhibition of PGE2 production and ~70% decrease of DPD activity. After 48 hours, the NSAIDs had more potently Control INDO NS-398 reduced the PGE2 level and also the DPD activity. (% of control) (% of control) Interestingly, the 10-day treatment did not further enhance the effect of NSAIDs (Table II). The 3-week treatment of the HCA-7 cells 24-hour animals with NSAIDs caused a significant decrease of the 1 PGE2 20.4±4.8 3.1±0.4 (15)* 3.6±0.1 (18)* PGE2 level and also of the DPD enzyme activity in HT-29 DPD activity2 101.3±15.6 36.0±1.4 (35)* 34.0±1.9 (34)* xenografts (Table II). The basal DPD activity of HCA-7 48-hour xenograft was also measured and proved to be relatively low PGE2 20.4±4.8 0.4±0.1 (2)* 0.2±0.1 (1)* (24±4.2 pmol/min/mg protein). DPD activity 101.3±15.6 18.1±1.1 (18)* 16.2±1.3 (16)* 10-day To ascertain that INDO and NS-398 are not direct DPD PGE2 20.4±4.8 0.4±0.1 (2)* 0.2±0.1 (1)* enzyme inhibitors, the DPD enzyme activity was measured DPD activity 101.3±15.6 18.1±0.9 (18)* 20.8±2.3 (21)* as described in our earlier study (15) by adding the NSAIDs HT-29 xenograft directly into the cytosol prepared from untreated HCA-7 3-week cells. No changes in DPD enzyme activity were detected. PGE2 2.8±0.1 0.4±0.3 (14)* 1.1±0.3 (39)* DPD activity 58.5±4.5 35±9 (60)* 17±7 (30)* DPD mRNA expression of HCA-7 cells after 24- and 48-hour 1ng/ml; 2pmol/min/mg protein. Data represent the mean±standard NSAID treatment. The DPD/GAPDH mRNA ratio of the deviation of independent experiments; *p<0.001 vs. control. control was 0.15. After 24-hour treatment with INDO, the ratio was 0.045 and that of NS-398 was 0.051. After 48-hour treatment, the ratios were 0.028 and 0.023 for INDO and NS- 398, respectively. All treatments resulted in a significant Statistical analysis. Statistical significance was determined by decrease of expression compared to the control. The DPD analysis of variance (ANOVA) and Tukey’s post hoc test using mRNA expressions were in correlation with the DPD activities. GraphPad PRISM software (San Diego, CA, USA). p-Values <0.05 were considered as significant. Uracil measurement of the cells after 24-hour NSAID treatment. To avoid a bias caused by eventual high endogenous Results DPD substrate levels, the intracellular concentration of uracil was measured. The endogenous uracil level was unchanged COX-2 protein expression. Xenografts were established from after NSAID treatments (INDO: 0.52±0.08 ng/106 cells, NS- both HT-29 and HCA-7 cell lines. In contrast with our 398: 0.53±0.06 ng/106) compared to the control (0.57±0.11 ng expectations based on our previous study (15), HT-29 proved /106 cells). to establish high, and HCA-7 low COX-2 expressing xenografts detected by specific anti-human antibody (Table I Discussion and Figure 1). Further studies are needed to understand these changes. Based on the summarized results of Table I, the 5-Fluorouracil is an effective cytotoxic agent in the treatments with NSAIDs were performed only on the high management of colorectal cancer, which after inhibiting COX-2-expressing HCA-7 cells and HT-29 xenografts. pyrimidine metabolism induces apoptosis of cancer cells. Previously it was determined that a 48-hour treatment of HCA- More than 80% of the administered 5-FU dose is degraded 7 cells with INDO and NS-398 non-significantly reduced the in vivo by DPD to 5,6-dihydro-5-fluorouracil (7). COX-2 expression (15). In the present investigation, a 10-day Combination of 5-FU with various modulators, e.g. DPD INDO or NS-398 treatment slightly but not significantly inhibitors, potentiated its antitumor effect and enhanced the reduced the COX-2 protein expression in HCA-7 cells (Figure rate of apoptosis (11, 12, 18). The level of DPD activity is 1). In HT-29 xenografts, the 3-week treatment with NSAIDs even a major determinant of the toxicity of 5-FU (7, 19). also resulted in a non-significant decrease of COX-2 protein Guimbaud et al. found that DPD activity was significantly expression (Figure 1). The results of immunofluorescent elevated in inflammatory diseases and tumor tissues of the staining of HCA-7 cells and immunohistochemistry of HT-29 colon compared to the normal epithelium (20). On the other xenografts correlated well with the above findings, showing hand, several studies have established that COX-2 that COX-2 expression in both cases remained practically overexpression is common in inflammation and also in unchanged even after a long-term NSAID treatment (Figure 2). human malignancies, including cancer of the colon. COX-2

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and translation of its DPYD are not well understood. Zhang et al. demonstrated that the Sp1 binds to the DPYD promoter and acts as a potent transactivator and induces DPYD promoter activity (22). COX-2 promoter is also under control through a tight regulatory network and is known to contain many trans-acting elements that bind a variety of transcription factors, including Sp1 (23). In addition, Wu et al. found that PGE2, the main product of COX-2, can also activate Sp1 (24). It is well known that COX-2 is the main target of NSAIDs. Interestingly, NSAIDs activate proteosome-dependent degradation of Sp1 (25). In this regard one can speculate that COX-2 and DPD might be connected via Sp1. NSAIDs have been recognised that can interfere with pentose phosphate pathways of glucose metabolism by inhibiting glucose-6-phosphate-dehydrogenase (G6PDH) and decreasing the level of NADPH (26), the of DPD in catabolising uracil to 5,6-dihydrouracil (5). The lower level of NADPH may also affect DPD activity. Furthermore, NSAIDs can influence the energy metabolism of the cells (27) by reducing the activity of the adenosine (ANT), which exchanges ADP for ATP (28); thus the lower level of ATP may also contribute to the lowering of the NADPH level. Many non-steroidal anti-inflammatory drugs were found to be competitive inhibitors of dihydrofolate (DHFR) (29), resulting in a deficiency causing a relative increase of the uridine monophosphate (UMP), the substrate Figure 3. Overview of the possible pathways and targets of NSAIDs. of (TS) enzyme. The high level of UMP 5-FU, 5-Fluorouracil; ADP, adenosine diphosphate; ANT, adenosine may induce its catabolism, increasing the uracil level (30). At nucleotide translocase; ATP, adenosine triphosphate; COX-2, the same time, Mashiyama et al. showed that folate cyclooxygenase-2; DHF, dihydrofolate; DHFR, DHF reductase; DPD, deficiency caused an accumulation of the cells in the S-phase dihydropyrimidine dehydrogenase; DPYD, DPD gene; G6PDH, glucose- of the cell cycle and increased uracil misincorporation into 6-phosphate-dehydrogenase; H2FU, dihydrofluorouracil; H2U, dihydrouracil; MTHF, methylenetetrahydrofolate; NADP/NADPH, DNA (31). In this way, the presumed increase of UMP nicotinamide adenine dinucleotide phosphate; NSAIDs, non-steroidal caused by might be re-equilibrated. These anti-inflammatory drugs; Sp1, specific protein 1; THF, tetrahydrofolate; findings are in agreement with our previous and present TMP, thymidylate monophosphate; TS, thymidylate synthase; UMP, results that treatment with low-dose NSAIDs caused S-phase uridine monophosphate. arrest and the endogenous uracil level remained unchanged after NSAID treatment. These results supported our finding that the decrease of DPD activity is not a bias caused by an increased endogenous uracil level. promotes tumor cell growth, angiogenesis, tumor invasion DPD activity has previously been reported to correlate with and metastasis (4, 21). its protein level in human lymphocytes (32). A correlation In the present study, the coexistence of COX-2 and DPD between the mRNA expression and catalytic activity of DPD activities was demonstrated and the effect of NSAIDs on these supports the relationship between its in vivo transcription and enzymes was studied in a COX-2-expressing human tumor translation (33). In our study, a similar correlation was found cell line and xenograft. Our study is the first to present a between DPD activity and its mRNA expression. The DPD simultaneous decrease of COX-2 and DPD activities in high mRNA expression of HCA-7 cells showed ~3- and 6-fold COX-2-expressing cells and xenografts treated with NSAIDs. decrease after 24- and 48-h INDO or NS-398 treatment, In order to understand our results regarding the synchronous respectively, compared to the control, which was in a good decrease of COX-2 and DPD activities and the inhibitory correlation with the reduction of DPD activity. effect of NSAIDs on these enzymes, a brief overview of the In conclusion, low-dose NSAID treatment simultaneously pathways and targets of NSAIDs is given (Figure 3). reduced both COX-2 and DPD activity in the high COX-2- Although DPD is a well-known biomarker of 5-FU-based expressing HCA-7 cell line and HT-29 xenograft without cancer chemotherapy, the regulatory mechanisms of transcription affecting endogenous uracil levels.

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According to the literary data, the cancer cell lines and 10 Saif MW, Syrigos KN and Katirtzoglou NA: S-1 a promising new oral fluoropyrimidine derivative. Expert Opin Investig xenografts can be characterized either by high PGE2 level Drugs 18: 335-348, 2009. and DPD activity, or by low PGE2 level and DPD activity. We could not found any cell line which produced great 11 Ahmed FY, Johnston SJ, Cassidy J, O’Kelly T, Binnie N, Murray GI, van Gennip AH, Abeling NG, Knight S and McLeod HL: amounts of PGE2 and had low DPD activity. In this regard, Eniluracil treatment completely inactivates dihydropyrimidine we raise the possibility that COX-2 and DPD are dehydrogenase in colorectal tumors. J Clin Oncol 17: 2439- coexpressed and/or regulated together. 2445, 1999. NSAIDs could be promising modulators of fluorouracil- 12 Kralovánszky J, Katona C, Jeney A, Pandi E, Noordhuis P, based chemotherapy, especially in high COX-2-expressing Erdélyi-Tóth V, Otvös L, Kovács P, Van der Wilt CL and Peters human cancer. It is worth mentioning that it was recently GJ: 5-Ethyl-2’-deoxyuridine, a modulator of both antitumour shown that combined therapy of fluoropyrimidines with action and pharmacokinetics of 5-fluorouracil. J Cancer Res Clin Oncol 125: 675-684, 1999. COX-2 inhibitors might hold promise for prophylaxis of 13 Ogino M and Hanazono M: Indomethacin preferentially liver metastasis of colorectal cancer (34). Further augments 5-fluorouracil cytotoxicity in Colon 26 tumors by investigations are still needed to understand the exact increasing the intracellular inflow of 5-fluorouracil. Int J Clin mechanism of the inhibition of DPD activity and thus the 5- Oncol 4: 22-25, 1999. FU catabolism by NSAIDs. 14 Ogino M and Minoura S: Indomethacin increases the cytotoxicity of cis-platinum and 5-fluorouracil in the human Acknowledgements uterine cervical cancer cell lines SKG-2 and HKUS by increasing the intracellular uptake of the agents. Int J Clin Oncol 6: 84-89, 2001. We thank Cs. Polényi Makácsné, A. Nagy, J. Kútvölgyi, A. Éber 15 Réti A, Barna G, Pap E, Adleff V, L Komlósi V, Jeney A, Mousáné, A. Sztodola and A. M. Borza for their technical assistance. Kralovánszky J and Budai B: Enhancement of 5-fluorouracil This study was supported by Jedlik Ányos Grant (NKFP1- efficacy on high COX-2 expressing HCA-7 cells by low-dose 00024/2005). indomethacin and NS-398 but not on low COX-2 expressing HT- 29 cells. Pathol Oncol Res 9126-9129, 2008. 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