Kaisa Sunela
email: kaisa.sunela@pshp.fitel.358331163082

Sanna Koskinen

Pirkko-Liisa Kellokumpu-Lehtinen

in locally advanced or metastatic renal cell cancer

Keywords: renal cell carcinoma, immunotherapy, interferon, pegylated, chemotherapy, capecitabine

A phase-II study of combination of pegylated interferon alfa-2a and capecitabine

Background

Until very recently, renal cell cancer (RCC) has had very few available oncological treatments with at least moderate efficacy. Interferon (IFN) has been reported to have antitumor activity against RCC already in 1983 producing partial responses (PR) to 26 % of patients in the early studies [START_REF] Quesada | Renal cell carcinoma: Antitumor effects of leukocyte interferon[END_REF]. The mechanisms of action of IFN are various and still partly unclear. IFN modulates cancer cell growth and differentiation by regulation of cell cycle, affects cellular communication and intracellular signaling making cells to undergo apoptosis, but also induces nonapoptotic cell death. The effects of IFN are translated in the direct tumour cell growth inhibition or death and in the stimulation of an antitumor immune response. It also upregulates expression of MCH class I antigens which favours tumor cell recognition by specific cytolytic T cells as well as the activation of natural killer cells. IFN has postgenomic effects based on the regulation of protein synthesis and on the selective translation of proteins participating in growth arrest and apoptosis. IFN also produces a link between innate and adaptive immune responses and interferes with tumourmediated angiogenesis. [START_REF] Tagliaferri | New pharmacokinetic and pharmacodynamic tools for interferon-alpha (IFN-alpha) treatment of human cancer[END_REF] However, IFN has moderate toxicity and the dosing is three to five times a week given as subcutaneous injections. Therefore new formulations have been developed and two pegylated interferons are now commercially available. These are chemically modified by covalent linkage of polyethylene glycol (PEG) polymer to enhance pharmacokinetic characteristics and reduce immunogenicity. [START_REF] Motzer | Phase I trial of 40-kd branched pegylated interferon alfa-2a for patients with advanced renal cell carcinoma[END_REF][START_REF] Talpaz | Phase 1 study of polyethylene glycol formulation of interferon-alfa-2B (Schering 54031) in Philadelphia chromosome-positive chronic myelogenous leukemia[END_REF] Pegylated interferon-alfa-2a (Pegasys®) belongs to the second generation of pegylated interferons, with a 40 kd PEG-moiety. After injection serum concentrations are measurable in six hours and stay close to peak from day three to eight allowing once-weekly dosing which might improve efficacy compared with non-pegylated interferons. Steady-state is seen from week five of therapy, but serum concentrations increase with chronic dosing to 2.5-fold. Side-effects are most commonly fatigue, fever, headache, myalgia, nausea and decreased appetite and elevated liver values, anemia, thrombocytopenia and leuko-/neutropenia. In phase I studies PRs were seen in 19 %, stable disease (SD) in 48%; 450 µg/week was recommend for further studies as a single-agent therapy. [START_REF] Motzer | Phase I trial of 40-kd branched pegylated interferon alfa-2a for patients with advanced renal cell carcinoma[END_REF] In phase II studies responses were seen in 13 % of patients, complete response (CR) in 2.5 %. The toxicity was mostly mild to moderate; grade 3-4 toxicity was seen as neutropenia, fatigue, nausea/ vomiting and elevated hepatic transaminase values. [START_REF] Motzer | Phase II trial of branched peginterferon-alfa 2a (40 kd) for patients with advanced renal cell carcinoma[END_REF] An alternative to pegylated interferon-alfa-2a is pegylated interferon-alfa-2b (PEG-Intron®), which has a single PEG-moiety and a molecular weight of 12 kd. As a single agent therapy it is well tolerated up to 6.0 µg/kg /week, which achieves an exposure comparable to 180 MIU non-pegylated interferon per week. [START_REF] Talpaz | Phase 1 study of polyethylene glycol formulation of interferon-alfa-2B (Schering 54031) in Philadelphia chromosome-positive chronic myelogenous leukemia[END_REF][START_REF] Bukowski | Pegylated interferon Alfa-2b treatment for patients with solid tumors: A phase I/II study[END_REF] In a phase II study with single agent therapy with escalating dose 3.0 to 6.0 µg/kg/week, the overall response rate was 13.6 %, with 4.5 % CRs (one patient). Median overall survival (OS) was 13 months; in 9 % of patients the response remained over six months. In this study fatigue was the major dose-limiting factor; in 22.7 % of patients grade 3-4 fatigue was reported. No grade 3-4 hematologic toxicity or liver enzyme elevations were seen, but 55 % of patients required dose reductions. [START_REF] Bex | A phase-II study of pegylated interferon alfa-2b for patients with metastatic renal cell carcinoma and removal of the primary tumor[END_REF] Chemotherapy is considered mostly ineffective in RCC [START_REF] Motzer | Systemic therapy for renal cell carcinoma[END_REF], but 5-fluorouracil as a continuous infusion has shown some (less than 10 %) PRs [START_REF] Kish | Evaluation of low dose continuous infusion 5-fluorouracil in patients with advanced and recurrent renal cell carcinoma. A Southwest Oncology Group Study[END_REF]. Capecitabine is an oral fluoropyrimidine, a prodrug which is converted to 5-fluorouracil via enzyme pathway. Kidney cancer cells contain a considerable amount of thymidine phosphorylase (TP), a key enzyme converting capecitabine to active 5-FU. [START_REF] Miwa | Design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver cancer tissue[END_REF] As a single-agent capecitabine gives minor responses even in third line therapy, but in most patients no response or only SD is observed [START_REF] Pagliaro | Phase II study of capecitabine singe-agent therapy in patients with metastatic renal cell carcinoma[END_REF][START_REF] Petrioli | Capecitabine as third-line treatment in patients with metastatic renal cell carcinoma after failing immunotherapy[END_REF].

The rationale for combination of capecitabine and interferon, is that cytokines, including interferon increase intratumoral levels of TP especially given prior to fluoropyrimides. This increases the efficacy of capecitabine. [START_REF] Eda | Cytokines induce thymidine phosphorylase expression in tumor cells and make them more susceptible to 5'-deoxy-5-fluorouridine[END_REF][START_REF] Morita | Biomodulation of 5-fluorouracil by interferon-alfa in human renal carcinoma cells: relationship to the expression of thymidine phosphorylase[END_REF] The continuous infusion of 5-fluorouracil, similar to the effect of capecitabine, is reported to potentiate the action of interferon-alfa-2b producing a 43 % response rate with 19 % CRs, with mild toxicity. These responses were durable as the mean duration of response was almost two years. [START_REF] Gebrosky | Treatment of renal cell carcinoma with 5-fluorouracil and alfa-interferon[END_REF] However, the response rates in multicenter randomized trials are similar to those for cytokines alone [START_REF] Negrier | Treatment of patients with metastatic renal carcinoma with a combination of subcutaneous interleukin-2 and interferon alfa with or without fluorouracil[END_REF].

Combinations of capecitabine and non-pegylated interferon-α has been tested in phase I and II studies, which have shown that this combination is most commonly well tolerated and it has some clinical activity and manageable toxicity [START_REF] Chang | Phase I trial of capecitabine in combination with interferon alpha in patients with metastatic renal cancer: toxicity and pharmacokinetics[END_REF][START_REF] Padrik | Combination therapy with capecitabine and interferon alfa-2A in patients with advanced renal cell carcinoma: a phase II study[END_REF][START_REF] Segota | Phase II trial of capecitabine and rHU-interferon-alfa-2a in patients with metastatic renal cell carcinoma, limited efficacy, and moderate toxicity[END_REF]. PRs and SD even as a second-or third-line treatment are seen with capecitabine as a single-agent therapy or in combination with interferon-alfa [START_REF] Wenzel | Capecitabine monotherapy and in combination with immunotherapy in the treatment of metastatic renal cell carcinoma[END_REF][START_REF] Wenzel | Capecitabine in the treatment of metastatic renal cell carcinoma failing immunotherapy[END_REF]. In triplet combinations immunomodulatory drug thalidomide is added to combination [START_REF] Amato | Interferon-alfa plus capecitabine and thalidomide in patients with metastatic renal cell carcinoma: A pilot study[END_REF]. Also combination of capecitabine, interferon-alfa-2a, IL-2 and oral 13-cis retinoic acid has been reported [START_REF] Oovermann | Capecitabine in the treatment of metastatic renal cell carcinoma[END_REF]. Only reported study on the combination of capecitabine (1000 mg/ m 2 twice daily days 1-14, cycle 28 days) and pegylated interferon-alfa (50 µg weekly s.c.) is done with PEG-Intron®. In this phase II study also subcutaneous low dose interleukin-2 (IL-2) and oral 13-cisretinoid acid were in the treatment schedule. This treatment was well tolerated; responses were seen in 53.6 % of patients. [START_REF] Recchia | Multicenter phase II study of chemo-immunotherapy in the treatment of metastatic renal cell carcinoma[END_REF] The combination of capecitabine and pegylated interferon-alfa-2a is not reported in the literature before this study.

Patients and methods

This was an opel-label, single institution, nonrandomized phase II study. Primary objective was to characterize efficacy, safety and tolerability of PEG-IFN-alfa-2a given once weekly s.c. and capecitabine given per orally. For efficacy progression-free survival (PFS), overall survival (OS) and response rate were assessed; for tolerability premature withdrawals and dose reductions because of laboratory abnormalities or adverse events and for safety adverse events, laboratory test results and vital signs were recorded. The study protocol was approved by the Pirkanmaa Hospital District ethical committee.

Eligibility criteria included: age 18 to 80 years, informed consent, histologically or cytologically verified locally advanced or metastatic RCC with or without prior nephrectomy, evaluable or measurable disease (RECIST), performance status of Zubrod 0-2 and adequate renal (serum creatinine less than 180 mmol/l) and hepatic function (bilirubin, aspartate aminotransferase (ASAT) and glutamyl transferase (GT) less than 1.5 times the upper limit of normal values).

Exclusion criteria included pregnant or lactating women, other malignancies than RCC excluding basal or squamous cell carcinomas of the skin and carcinoma in situ of the uterine cervix, metastases in central nervous system (brain computed tomography was not required for asymptomatic patients), chronic heart failure (New York Heart Association status III-IV), instable angina pectoris, acute myocardium infarction within the past 12 months before study entry, history of difficult renal, hepatic, neurological or psychiatric disease, untreated depression, serious infection or wide-field radiotherapy within the previous four weeks. Prior treatment with interferon was not allowed.

Pretreatment evaluation included a medical history (including primary TNM classification and grade, sites of metastases, type of surgery, given radiotherapy), clinical examination, electrocardiography, complete blood count, assessment of plasma creatinine, calcium and protein levels and liver function tests: alkaline phosphatase (AFOS), alanine aminotransferase (ALAT), ASAT, GT, bilirubin. Radiographic studies (bone scan, thorax x-ray and abdominal ultrasound or whole body computed tomography) were assessed within 14 days prior to the initiation of therapy. In six first patients laboratory studies were performed on a weekly basis during the first three cycles, and at the end of each cycle thereafter. Radiographic studies, as required for assessment of measurable disease, were performed after two cycles, then every three cycles thereafter. Response was assessed according to RECIST criteria [START_REF] Therasse | New guidelines to evaluate the response to treatment in solid tumors[END_REF]. No external verification of response was employed. Toxicity was assessed according to NCI-CTCAE criteria [26] and quality of life according to EORTC QLQ-3 criteria [START_REF] Aaronson | The European Organisation for Research and Treatment of Cancer QLQ-C30: A quality-of-life instrument for use in international clinical trials in oncology[END_REF], both in base line and after every cycle. Treatment was continued until 52 weeks or clinically significant progression, patient's withdrawal, unacceptable toxicity or 12 weeks after confirmation of CR.

Between 2003 and 2008, 26 patients with advanced RCC entered trial (demographics in Table 1). The median age of patients was 63.5 years (range 46-81). One patient was older than the inclusion criteria required. However, this patient tolerated the treatment well and achieved complete response. Two patients were not operated before this trial.

PEG-IFN-alfa-2a was administered by subcutaneous injection at a fixed dose of 180 µg once weekly. As the study combination is not reported in the literature, PEG-IFN dosing was chosen to be the smallest with clinical efficacy [START_REF] Motzer | Phase I trial of 40-kd branched pegylated interferon alfa-2a for patients with advanced renal cell carcinoma[END_REF]. No dose modifications were done, but the injection was withheld for grade 3-4 toxicity and for grade 2 toxicity if it was related to interferon. Capecitabine was administered twice daily at a dose of 2000 mg/m 2 per day on days 1-14 of a 21-day cycle. Treatment was withheld in the event of any toxicity with severity exceeding grade two. With grade one toxicity, no delays or dose modifications were done. With grade two toxicity, treatment was withheld until toxicity improved to grade one or better, if the event was not treatable with symptomatic medication. If the same event reappeared, treatment was again withheld until toxicity improved to grade one or better, then restarted with 25 % lower dose of capecitabine. Grade three toxicity lead to withhold of therapy until it resolved to grade one or better, then restarted with 25 % lower dose of capecitabine. The reductions were made with 500 mg steps (the tablet size). As a dose reduction was recorded the lower dose of given therapy or shorter administration of capecitabine or omitted injection. Grade four toxicity lead to discontinuation of the therapy.

The follow-up was every two months until six months after the end of the study treatment and then every three to six months up to two years. The progression date and the cause and date of death were recorded.

The investigators designed and initiated the study, collected, analyzed and maintained the data. PEF-IFN (Pegasys®) and capecitabine (Xeloda®) were received from Roche Finland and grants from the Competitive Research Funding of Tampere University Hospital, Pirkanmaa Hospital District.

Results

The median of given cycles was 5.5 (range 1-18). In 30 % of patients the treatment discontinued before the third cycle because of toxicity or progressive disease. The median follow-up was 10.5 months (range 0-50 months).

The overall response rate was 27 %: PR was achieved in 23 % and CR in one patient (4%). SD was seen in 42 % of patients and progressive disease in 15 %. No response evaluations were done in 4 (15 %) patients because of toxicity related early discontinuation of the study treatment. In the end of study treatment, PR remained in 15 % and SD in 27 %, others progressed. CR remained throughout the follow-up period. The median PFS was 7.5 months (range 1-51+ months). Four patients have not progressed in the follow-up. The median OS was 17 months (range 2-55+ months). The oneyear survival was 77 %; the two-year survival rate was 38 %. Kaplan-Meier estimates of overall and progression-free survival are shown in Fig. 1. Second-line treatment was given to 72 % of patients.

In seven responders (CR + PR) the median age was 66 (range 49-81). Previous nefrectomy was performed to 86 % of these patients. Single site of metastases was seen in 86 % and only pulmonary metastases in 43 %. Primarily metastatic disease was noticed in 43 % of responders. One patient with CR had T2N0M0 grade IV disease operated six months before relapse (abdominal lymph nodes) was noticed.

The clinical toxicity of the study combination is shown in Table 2, percentages are shown per patient (n=26) and per given cycle (n=183): grade 1-2 and grade 3-4 toxicities are combined. Fatigue and nausea were the most common toxicities. Only 12 % of patients did not experience nausea at all and 4 % did not experience fatigue. When counted per cycle, in 66 % of given cycles patients experienced fatigue of any grade, nausea of any grade was seen in 33 % of the treatment cycles. Hand-foot syndrome was seen in 39 % of cycles, but 83 % of the patients did not experience this toxicity. The grade 3-4 toxicity (% of patients) was recorded on fatigue (23 %), nausea (12 %), constipation (4 %), diarrhea (15 %), stomatitis (4 %) and hand-foot syndrome (4 %). Also one treatment-related pulmonary embolus and one hematoma of the gall bladder were recorded (grade 4), one patient experienced grade 3 dyspnea. No treatment related deaths occurred. Other, miscellaneous toxicity of any grade was experienced by 77 % of patients during the study treatment; mostly these were grade 1-2. The following toxicities were listed: thrombophlebitis, decreased appetite, nail problems, dryness of mouth, abdominal pain, conjunctivitis and blurriness of the vision.

The hematological and blood chemistry toxicity is shown in Table 3, percentages are shown again per patient and per given cycle. The most common toxicities were anemia, leucopenia, neutropenia, hypoalbuminemia and the elevation of liver values (ASAT). Leucopenia was seen in 69 % of patients, anemia in 65 % and elevation of liver values were seen in 62 %. Anemia was also the most common toxicity when counted per given cycle: in 54 % of cycles. Neutropenia and thrombocytopenia were seen in 46 % and hypoalbuminemia in 45 % of cycles. Grade 3 -4 toxicity was seen as neutropenia, leucopenia and elevation of liver values.

Altogether 46 % of patients experienced grade 3-4 toxicity. However, in 93 % of cycles patients experienced only mild or no toxicity. Three patients (11.3 %) experienced grade 4 toxicity which lead to discontinuation of the study treatment: bloody diarrhea in two patients and synchronous pulmonary embolus and hematoma of the gall bladder in one patient.

In the evaluation of quality of life patients experienced the physical and psychological well being stable (Fig. 2). There was no statistically significant difference in the quality of life when tested in base line and in cycles five and ten (p=0.76).

The dose reductions are shown in Table 4. The most common indications for dose reductions were elevation of liver values and neutropenia. No dose reductions were needed in 15 % of patients and in 39 % of cycles. In 50 % of patients and in 39 % of given cycles was needed one dose reduction. Altogether 64 % of dose reductions were because of abnormal laboratory values (anemia, leuco-or neutropenia, thrombocytopenia, elevated liver values) which did not cause any clinical problems, but delayed the next cycle. One patient needed dose reduction five times, but continued in the study because of clinical benefit. In some cases there were multiple reasons to dose reductions. Dose delays were needed in 46 % of patients and in 14 % of given cycles. These delays were one to two weeks in duration. The dose intensity of capecitabine was 90-100 % in 40 % of cycles, 70-79 % in 36 % of cycles and 50-59 % in 14 % of cycles. Some injections of pegylated interferon were omitted in 35 % of patients and in 87 % of cycles because of grade 2-3 hematological toxicity or elevated liver values.

The reason for discontinuation of the treatment was toxicity in 11 patients (38 %), progressive disease in 10 (35 %) and non-treatment-, non-cancer-related death in one (3 %). In three patients (11.5 %) treatment was given up to one year and according to protocol discontinued then. One patient discontinued the treatment in complete remission 12 weeks after the confirmation of the response. Also congestive heart failure, infection and second malignancy were reasons for discontinuations, each in one patient. Three patients had two different reasons for discontinuation. Those who discontinued the study because of serious clinical side effects did it during the first cycles, after that there were mostly elevated liver values which prevented the continuation of the study treatment.

Discussion

The study combination with capecitabine and pegylated interferon-alfa-2a was active: 27 % of patients achieved response including 4 % of CRs. Most patients who responded here had only one site of metastases and half of them had only pulmonary metastases. Other single sites were abdominal and mediastinal lymph nodes. Typical responders to IFN are patients with only pulmonary metastases [START_REF] Quesada | Renal cell carcinoma: Antitumor effects of leukocyte interferon[END_REF]. The current combination proved efficacy also in other patients. The age range of responders was wide and even the oldest patient (81 years) tolerated the treatment well.

The achieved response rate is in line with phase II studies done with the combination of capecitabine and non-pegylated interferon: 12 to 24 % overall response rates are reported, with 3-4 % CRs; SD was seen in 36-38 % of patients [START_REF] Padrik | Combination therapy with capecitabine and interferon alfa-2A in patients with advanced renal cell carcinoma: a phase II study[END_REF][START_REF] Segota | Phase II trial of capecitabine and rHU-interferon-alfa-2a in patients with metastatic renal cell carcinoma, limited efficacy, and moderate toxicity[END_REF]. The triplet combination with thalidomide does not produce better responses: PRs were seen in 20 %, SD in 28 % [START_REF] Amato | Interferon-alfa plus capecitabine and thalidomide in patients with metastatic renal cell carcinoma: A pilot study[END_REF]. When IL-2 is added to combination [START_REF] Oovermann | Capecitabine in the treatment of metastatic renal cell carcinoma[END_REF][START_REF] Recchia | Multicenter phase II study of chemo-immunotherapy in the treatment of metastatic renal cell carcinoma[END_REF] more responses and longer PFS and OS are achieved. With the four-drug combination with IL-2 and 13-cis retinoic acid is reported response rate of 34 %, with 7 % CRs and 40 % of patients presented with SD [START_REF] Oovermann | Capecitabine in the treatment of metastatic renal cell carcinoma[END_REF]. Again, in the phase II study done with capecitabine, pegylated interferon-alfa-2b, IL-2 and 13-cis retinoic acid is reported responses in 53.6 % of patients, with 24 % CRs. SD was achieved in 34 %. The median PFS was 14.7 months and the median OS 27.9 months. [START_REF] Recchia | Multicenter phase II study of chemo-immunotherapy in the treatment of metastatic renal cell carcinoma[END_REF] When capecitabine is combined with another chemotherapy agent gemcitabine responses are seen in 15.8 %-22 % of patients in phase I-II studies [START_REF] Rini | A phase I trial of fixed dose rate gemcitabine and capecitabine in metastatic renal cell carcinoma[END_REF][START_REF] Waters | Phase II clinical trial of capecitabine and gemcitabine chemotherapy in patients with metastatic renal carcinoma[END_REF], which is quite comparable to responses in different combinations of chemoimmunotherapy with interferon.

Cytokines have been tested in combinations without chemotherapy regimens in hope for achieving more efficacy. When PEG-interferon is combined with IL-2 alone, the response rate is 30.2 % with 3.8 % CRs, which is comparable to the four-drug combinations [START_REF] Oovermann | Capecitabine in the treatment of metastatic renal cell carcinoma[END_REF][START_REF] Recchia | Multicenter phase II study of chemo-immunotherapy in the treatment of metastatic renal cell carcinoma[END_REF] or high dose IL-2 alone. The median duration of response was 11 months. This schedule consisted of PEG -IFN-2b 2.0 µg/kg/week and IL-2 5 MIU/m 2 x3 day 1, then once daily five days a week, both for four weeks followed by two weeks rest. [START_REF] Clark | Phase I/II trial of outpatient PEG-interferon with interleukin-2 in advanced renal cell carcinoma: a Cytokine Working Group Study[END_REF] In other schedule the pegylated interferon-alfa-2b was given 3.0 µg/kg/weekly and IL-2 20 MIU/m 2 three times a week at weeks 1 and 4, and 5.0 MIU/m 2 three times a week during weeks 2-3 and 5-6. PRs were seen only in 15 % of patients, SD was achieved in 68 %. PFS was 9.0 months and OS 31.9 months. [START_REF] George | Phase I trial of PEG-interferon and recombinant IL-2 in patients with metastatic renal cell carcinoma[END_REF] Toxicity of the study combination was manageable, but grade 3-4 side effects were seen in 46 % of patients. Dose reductions were needed in 85 % of patients and in 39 % of cycles. Despite of reductions the response remained in most patients. This need to reductions has been seen already in previous studies done with non-pegylated interferon. In the first phase I trial the combination of capecitabine (825-1000 mg/m 2 twice daily for 14 days, followed by one week rest) and non-pegylated interferon-alfa-2b (1.5-3.0 MIU/m 2 three times a week) had moderate toxicity, mostly mild nausea/ vomiting, diarrhea and hand-foot syndrome. The majority of patients experienced malaise, fever and chills related to interferon. Grade one hepatic toxicity occurred infrequently and hematologic toxicity was generally mild: thrombocytopenia was seen in 33 % of patients and neutropenia in 37 %. [START_REF] Chang | Phase I trial of capecitabine in combination with interferon alpha in patients with metastatic renal cancer: toxicity and pharmacokinetics[END_REF] Since then, in phase II study done with capecitabine (1250 mg/m 2 twice daily for 14 days followed by one week rest) and non-pegylated IFN (6 MIU x 3 /week) grade 3-4 toxicities were seen in 48 % including fatigue, nausea, hand-foot syndrome, anorexia, vomiting, anemia and neutropenia. Toxicity was considered acceptable, but dose reductions had to do in 48 % and dose delays were necessary in 16 % of patients. Capecitabine dose of 1000 mg/m 2 twice daily was recommended to further studies. [START_REF] Padrik | Combination therapy with capecitabine and interferon alfa-2A in patients with advanced renal cell carcinoma: a phase II study[END_REF] In another phase II study the starting dose with capecitabine was 1000 mg/m 2 twice daily on days 1-14, but because of toxicity requiring dose reductions during the first cycle the dose was reduced to 825 mg/m 2 twice daily.

was given 3.0 MIU/m 2 thrice a week. Toxicity was considered moderate to severe; dose reductions were required in 88 % of patients and 19 % of patients discontinued therapy secondary to toxicity. Grade 3-4 toxicity was seen in 78 %, most commonly gastrointestinal and hand-foot syndrome. [START_REF] Segota | Phase II trial of capecitabine and rHU-interferon-alfa-2a in patients with metastatic renal cell carcinoma, limited efficacy, and moderate toxicity[END_REF] In the study of combination of capecitabine and pegylated interferon-alfa-2b grade 3-4 toxicity was seen in 21.9 % including nausea and vomiting, neutropenia, anemia and thrombocytopenia. [START_REF] Recchia | Multicenter phase II study of chemo-immunotherapy in the treatment of metastatic renal cell carcinoma[END_REF] In the future, in this combination with pegylated interferon-alfa-2a the dose level for capecitabine should be 1600 mg/m 2 per day as the dose intensity over 70 % was here tolerable in 81 % of cycles (Table 4).

In the triplet combination (capecitabine 1900 mg/m 2 /day days 1-14; cycle 21 days, IFN-α 1 MIU daily without interruptions and thalidomide 400 mg daily) the dose reductions of capecitabine were required in 76 % of patients. The most common grade 3-4 toxicity was hand-foot syndrome; anemia occurred in 4 %, but no other grade 3-4 hematological toxicity was seen. [START_REF] Amato | Interferon-alfa plus capecitabine and thalidomide in patients with metastatic renal cell carcinoma: A pilot study[END_REF] In the four-drug combination of capecitabine, interferon-alfa-2a, IL-2 and oral 13-cis retinoic acid toxicity was mild. [START_REF] Oovermann | Capecitabine in the treatment of metastatic renal cell carcinoma[END_REF] When capecitabine is combined with gemcitabine, the combination is reported to be too toxic in phase I study despite of dose reductions [START_REF] Rini | A phase I trial of fixed dose rate gemcitabine and capecitabine in metastatic renal cell carcinoma[END_REF], but in phase II study this combination had moderate and manageable toxicity [START_REF] Waters | Phase II clinical trial of capecitabine and gemcitabine chemotherapy in patients with metastatic renal carcinoma[END_REF], even when gemcitabine was given with bigger dose than in the phase I study.

Toxicity of pegylated interferon-alfa seems to be schedule dependent and sometimes unpredictable. Also, it seems that two available pegylated interferons have different toxicity scale. This is probably in correlation with molecular weight. A 40-kd PEG moiety of interferon-alfa-2a cannot be excreted by the kidneys and is not quickly metabolized; therefore the PEG may accumulate in the liver, which increases the risk for toxicity especially in the long-term administration [START_REF] Bukowski | Pegylated interferon Alfa-2b treatment for patients with solid tumors: A phase I/II study[END_REF]. The combination of pegylated interferon-alfa-2a and IL-2 produces in some schedules inacceptable toxicity: grade 3-4 cardiac toxicity was seen in 17 % and central nervous system events in 11.3 %. The schedule was abandoned because of the toxicity, which remained unexplained as the serum levels of either of the drugs were not increased. Some pharmacodynamic effect was suspected; capillary leak by IL-2 may have increased the end organ concentrations. [START_REF] Clark | Phase I/II trial of outpatient PEG-interferon with interleukin-2 in advanced renal cell carcinoma: a Cytokine Working Group Study[END_REF] In other schedule toxicity was moderate; the most common toxicities were nausea and vomiting, diarrhea, fatigue, fever, transient elevation in liver transaminases, leucopenia, thrombocytopenia and anemia. Most of these side effects were mild. [START_REF] George | Phase I trial of PEG-interferon and recombinant IL-2 in patients with metastatic renal cell carcinoma[END_REF] Current treatment guide lines are recommending tyrosine kinase inhibitors (TKI), combination of bevacizumab and non-pegylated interferon or temsirolimus as primary choices to the first line treatment of metastatic RCC. The choice should be done according to tolerability of the treatment and prognostic factors. [32] With the combination of VEGF inhibitor bevacizumab and non-pegylated IFN, PFS was 10.2 months, OS 19.9 months and response rate 31 %. This combination is usually well tolerated, but grade 3-4 fatigue, diarrhea, anemia, neutropenia and thrombocytopenia are seen. Proteinuria, thromboembolic events and gastrointestinal perforation are additional side effects concerning treatment with bevacizumab. [START_REF] Escudier | Bevacizumab plus interferon alfa-2a for treatment of metastatic renal cell carcinoma: a randomised, double-blind phase III trial[END_REF] TKI sunitinib has proved efficacy in the first line treatment with response rate of 47 %, PFS of 11 months and OS of 26.4 months. As the current combination, sunitini b causes grade 3-4 diarrhea, fatigue, nausea and vomiting and hand-foot -syndrome, but additionally also hypertension. Grade 3 leuco-and neutropenia, thrombocytopenia and elevated liver values are also seen. [START_REF] Motzer | Sunitinib versus interferon alfa in metastatic renal-cell carcinoma[END_REF][START_REF] Motzer | Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma[END_REF] Temsirolimus is indicated to RCC patients with poor prognosis: median OS of 10.9 months and PFS of 5.5 months are reached. It also causes grade 3-4 hematological side effects and asthenia. Particular side effects concerning this treatment are hyperlipidemia, hyperglycemia and pneumonitis. [START_REF] Hudes | Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma[END_REF] The current study combination did not show any new side effects which were not known from the single agent studies. We did not select patients according to risk factors, but the achieved response rate, PFS and OS were better than with temsirolimus but lower than with sunitinib or with the combination of bevacizumab and interferon.

As pegylated IFNs are more convenient to use than non-pegylated IFN, they might find their place and more regular use in the treatment of RCC patients in combination with bevacizumab or in the second line treatment after TKIs in the current combination. Usually, TKIs are more tolerable than this study combination but with lower starting dose of capecitabine the situation can be different. With lower dose of capecitabine most of the clinically significant grade 4 side effects are probably prevented. As the clinical benefit was clear, we recommend a phase-III study to clarify the position of the current study combination.

Conclusions

The combination of capecitabine and pegylated interferon-alfa-2a produced clinical benefit of 69 %, including 23 % PRs and 4 % CRs. Three responders had only pulmonary metastases and three had lymph node metastases, one patient had multiple sites. However, the response rate was not better than in studies including non-pegylated interferon. The median PFS was 7.5 months and OS 17 months. Dose reductions were needed in 85 % of patients and dose delays in 46 %, which is in line with previous studies. Toxicity was still manageable, but because of many reductions the dose for capecitabine should be 1600 mg/m 2 per day in the future studies. The most common toxicities were fatigue, nausea, hematological toxicities and elevated liver values. Because of more convenient dosing, the pegylated interferon-alfa-2a would be useful to be studied in combination with bevacizumab. The current combination might be applicable in the second line treatment after TKIs, but a phase-III study is still needed. At the moment, patients unsuitable to treatment with sunitinib or bevacizumab (such as hypertensive patients or cases with previous arterial thromboembolic event or gastrointestinal perforation) could be candidates for this combination.

Fig. 1 .

 1 Fig. 1. Kaplan-Meier estimates of overall and progression-free survival.

Table 1 .

 1 Patient demographics

	Demographics

Table 2 .

 2 Clinical toxicity

	Toxicity	Grade	Per patient n (%)	Per cycle n (%)
	Fatigue	0	1	(4)	62	(34)
		1-2	19	(73)	115	(63)
		3-4	6	(23)	6	(3)
	Muscle pain	0	19	(73)	148	(81)
		1-2	7	(27)	35	(19)
		3-4	0	(0)	0	(0)
	Fever	0	13	(50)	153	(84)
		1-2	13	(50)	30	(16)
		3-4	0	(0)	0	(0)
	Neuropathy	0	17	(65)	157	(86)
		1-2	9	(35)	26	(14)
		3-4	0	(0)	0	(0)
	Constipation	0	12	(46)	156	(85)
		1-2	13	(50)	26	(14)
		3-4	1	(4)	1	(1)
	Stomatitis	0	15	(58)	161	(88)
		1-2	10	(38)	21	(11)
		3-4	1	(4)	1	(1)
	Nausea and	0	3	(12)	124	(68)
	vomiting	1-2	20	(77)	56	(31)
		3-4	3	(12)	3	(2)
	Diarrhea	0	12	(46)	157	(86)
		1-2	10	(38)	22	(12)
		3-4	4	(15)	4	(2)
	Infection	0	13	(50)	160	(87)
		1-2	13	(50)	23	(13)
		3-4	0	(0)	0	(0)
	Alopecia	0	21	(81)	171	(93)
		1-2	5	(19)	12	(7)
		3-4	0	(0)	0	(0)
	Rash	0	13	(50)	146	(80)
		1-2	13	(50)	37	(20)
		3-4	0	(0)	0	(0)
	Allergic symptoms	0	26	(100)	183	(100)
		1-2	0	(0)	0	(0)
		3-4	0	(0)	0	(0)
	Hand-foot syndrome 0	15	(58)	112	(61)
		1-2	10	(38)	70	(38)
		3-4	1	(4)	1	(1)
	Other symptoms	0	6	(23)	130	(71)
		1-2	18	(69)	51	(28)
		3-4	2	(8)	2	(1)


Andrea Réti

Éva Pap

Vilmos Adleff

András Jeney

Dr Judit Kralovánszky

Barna Budai

Enhanced 5-fluorouracil cytotoxicity in high cyclooxygenase-2 expressing colorectal cancer cells and xenografts induced by non-steroidal anti-inflammatory drugs via downregulation of dihydropyrimidine dehydrogenase

Keywords: indomethacin, NS-398, 5-fluorouracil, cyclooxygenase-2, dihydropyrimidine dehydrogenase

Introduction

One of the most widely used anticancer agents for treatment of colorectal cancer is 5-FU. The rate-limiting enzyme of 5-FU catabolism is dihydropyrimidine dehydrogenase (DPD).

Besides the liver and the peripheral mononuclear cells, tumoral DPD has become of clinical interest because elevated intratumoral DPD can decrease the tumor response to 5-FU therapy since more than 80% of the administered 5-FU is catabolised in vivo by DPD to 5,6-dihydro-5-fluorouracil. The degradation of 5-FU reduces its efficiency and requires extremely high doses and the fluorinated products may cause side-effects in the nervous system [START_REF] Schnackerz | Dihydropyrimidine dehydrogenase: a flavoprotein with four iron-sulfur clusters[END_REF].

In order to improve the effectiveness of 5-FU, a new subclass of orally administrated fluoropyrimidines, UFT and S1, containing potent DPD inhibitors, i.e: uracil and 5-chloro-2,4 dihydroxypyridine (CDHP), respectively, were developed [START_REF] Hoff | The tegafur-based dihydropyrimidine dehydrogenase inhibitory fluoropyrimidines, UFT/leucovorin (ORZEL TM ) and S-1: a review of their clinical development and therapeutic potential[END_REF]. An irreversible inhibitor of DPD, ethynyluracil, was also studied and found to be effective in clinical trials [START_REF] Ahmed | Eniluracil treatment completely inactivates dihydropyrimidine dehydrogenase in colorectal tumors[END_REF]. Kralovánszky et al. proved that 5-ethyl-2'-deoxyuridine enhanced the therapeutic index of 5-FU by reducing its catabolism [START_REF] Kralovánszky | 5-Ethyl-2'-deoxyuridine, a modulator of both antitumour action and pharmacokinetics of 5-fluorouracil[END_REF].

Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme in the synthesis of prostanoids and is common in human malignancies, including cancer of the colon. The upregulat ion of COX-2 promotes tumor cell growth, angiogenesis, tumor invasion and metastasis. Non-steroidal antiinflammatory drugs (NSAIDs) inhibit the COX-2 enzyme activity. Several preclinical and clinical studies have shown that NSAIDs are effective chemopreve ntive and antitumor agents either alone or in combination with standard cancer therapies [START_REF] De Groot | Non-steroidal anti-inflammatory drugs to potentiate chemotherapy effects: from lab to clinic[END_REF][START_REF] Bennett | Studies on the mechanism by which indomethacin increases the anticancer effect of methotrexate[END_REF][START_REF] Totzke | Cyclooxygenase-2 (COX-2) inhibitors sensitize tumor cells specifically to death receptor-induced appoptosis independently of COX-2 inhibition[END_REF]. Previously it was shown that NSAIDs, indomethacin and NS-398, applied in low dose effectively increased the 5fluorouracil (5-FU)-sensitivity in high COX-2 protein-expressing HCA-7 colon cancer cells [START_REF] Réti | Enhancement of 5-Fluorouracil Efficacy on High COX-2 Expressing HCA-7 Cells by Low-Dose Indomethacin and NS-398 but not on Low COX-2 Expressing HT-29 Cells[END_REF]. In a recent report from our laboratory it was also demonstrated that two NSAIDs:

Materials and methods

Cell lines

HT-29 and HCA-7 cells were purchased from the European Collection of Cell Cultures (Salisbury, UK) and were cultured in Dulbecco's modified Eaglemedium and RPMI, respectively, supplemented with 10 % (v/v) fetal bovine serum (Sigma-Aldrich, St. Louis, USA).

Coating of plates or glass coverslips

Six-and 96-well plates or glass coverslips coated with 10 μg/ml human collagen type IV, fibronectin or laminin (Sigma-Aldrich, St. Louis, USA) were incubated for 1 h at 37ºC then washed with PBS. The HT-29 cells were seeded on the coated plates and incubated for 4 hours.

Immunofluorescence staining of COX-2 and DPD proteins

The HT-29 cells were incubated for 4 hours on glass coverslips coated with ECM components. Then cells were fixed in methanol at -20°C for 10 min. and washed twice with PBS. Monolayers were incubated with COX-2 (Cayman Chemicals, Ann Arbor, MI, USA) or DPD (Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary monoclonal antibodies (1:100 and 1:50, respectively) in Superblock Blocking Buffer in TBS for overnight at 4°C. Following PBS washes monolayers were incubated with the FITC conjugated secondary antibodies (1:1000, respectively) (Alexa Fluor 488, Invitrogen, USA) in Superblock Blocking Buffer in TBS for 2 h at 4 C. After further PBS washes the fluorescence was detected by Olympus CKX41 fluorescence microscope (Tokyo, Japan) and documented with Olympus C-5060 widezoom camera (Tokyo, Japan).

Prostaglandin E 2 assay

The HT-29 cells (1x10 6 ) in 1ml culture media were seeded onto non-coated or collagen IV coated wells of 6-well plates. After 4 hours conditioned media were harvested and the concentration of prostaglandin (PGE 2 ) was measured by ELISA (Assay Designs, Ann Arbor, MI, USA) according to the manufacturer's protocol. Measurements were made in triplicate in three separate experiments.

Determination of cell growth inhibition

The uninduced and collagen-induced HT-29 cells grown in 96-well plates were treated with 10 μM 5-FU [START_REF] Réti | Enhancement of 5-Fluorouracil Efficacy on High COX-2 Expressing HCA-7 Cells by Low-Dose Indomethacin and NS-398 but not on Low COX-2 Expressing HT-29 Cells[END_REF] alone or combined with non selective COX-2 inhibitor, indomethacin (Sigma-Aldrich, St. Louis, USA) or selective COX-2 inhibitor, NS-398 (Cayman Chemicals, Ann Arbor, USA) (10 and 1.77 M, respectively) for 8 hours. The sulphorhodamine B (SRB) colorimetric assay was used to determine the cell growth inhibitory effect of the drugs in 96-well plates. The detailed SRB assay was described earlier [START_REF] Réti | Enhancement of 5-Fluorouracil Efficacy on High COX-2 Expressing HCA-7 Cells by Low-Dose Indomethacin and NS-398 but not on Low COX-2 Expressing HT-29 Cells[END_REF]. The experiments were done in six wells of the 96-well plates and repeated three times.

Treatment of collagen-induced HT-29 (HT-29-C) and HCA-7 cells for determination of DPYD mRNA expression and enzyme activity

Collagen-induced HT-29 cells grown in 6-well plates were treated for 8 hours with 5-FU alone or combined with indomethacin or NS-398 as described above. HCA-7 cells grown in 6-well plates were treated with 1 mM 5-FU [START_REF] Réti | Enhancement of 5-Fluorouracil Efficacy on High COX-2 Expressing HCA-7 Cells by Low-Dose Indomethacin and NS-398 but not on Low COX-2 Expressing HT-29 Cells[END_REF] alone or combined with indomethacin or NS-398 (10 and 1.77 M, respectively) for 24 hours.

Total RNA extraction and cDNA synthesis HT-29-C and HCA-7 cells grown in 6-well plates were harvested and total RNA was isolated using RNeasy mini prep kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. The concentration of RNA was determined by measuring the absorbance at 260 nm with spectrophotometer (BIO-TEK Instruments, VM, USA). Reverse-transcription was performed using RevertAid M-MuLV reverse transcriptase (Fermentas, Glen Burnie, MD, USA). The mRNA templates (2 μg) were transcribed with 0.5 μg oligo (dT) primer (Fermentas) according to the manufacturer's protocol.

Quantitative real-time RT-PCR

The DPYD and COX-2 mRNA expressions were determined by real time RT-PCR. The βactin (ACTB) gene was used as reference. The ACTB primers were: 5'-GTGGGGCGCCCCAGGCACCA and 5'-CTCCTTAATGTCACGCACGATTTC. The primers used for amplification of DPYD were: 5'-GAAATGGCCGGATTGAAGTTT and 5'-TCGAAGAGCTTTTGAAGCTGG and that for COX-2 were: 5'-TTCAAATGAGATTGTGGGAAAATTGCT and 5'-AGTTCATCTCTGCCTGAGTATCTT.

The amplifications were performed in a mixture of 1 μl cDNA, 0.2 μl o f primers, 5 μl IQ SybrGreen Supermix (Bio-Rad, Hercules, CA, USA) and 3.6 μl H 2 O on a RotorGene 2000 machine (Corbett Research, Mortlake, Australia). The 3 min denaturation at 95ºC was followed by 40 cycles of 20 s denaturation at 95ºC, 20 s annealing at 62ºC and 30 s extension at 72ºC. The amplicon purity was checked by melting curve analysis. The relative expression levels were calculated as described by Pfaffl [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]. The results are presented as mean of three experiments.

HT-29 xenografts

A suspension of 2 10 6 HT-29 cells in physiological saline (0.1 ml) was injected subcutaneously into the hind leg of 6-week-old female SCID mice. All animal experiments were performed according to the ethically approved guidelines of the Animal Care Facility of the 1 st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Budapest.

The animal weight (g) and tumor diameters (mm) were measured before the treatment and on the 2 nd , 4 th and 6 th days with calliper and the tumor volume (mm 3 ) was calculated as follows:

π x (shortest diameter) 2 x (longest diameter)/6.

Treatment of xenograft-bearing mice

The HT-29 tumor bearing mice were randomly separated into 7 groups, 4 mice each: vehicle control, 6 or 30 mg/kg/day 5-FU and 6 or 30 mg /kg/day 5-FU + indomethacin or NS-398.

The 5-FU (Pharmachemie, Haarlem, Hollandia) was given in two doses: 6 and 30 mg/kg body weight in 0.2 ml saline and was administered by i.p. injection for 5 days. The applied 30 mg/kg x 5 days 5-FU was regarded as the maximal tolerated dose [START_REF] Guichard | Sequence-dependent activity of the irinotecan-5FU combination in human colon-cancer model HT-29 in vitro and in vivo[END_REF].

The dose of indomethacin and NS-398 was 2.5 and 5 mg/kg, respectively, in 0.2 ml saline and given by oral gavage for 5 days. These doses are qualified as relatively low doses [START_REF] Eli | Comparative effects of indomethacin on cell proliferation and cell cycle progression in tumor cells grown in vitro and in vivo[END_REF][START_REF] Matsuo | Inhibition of human glioma cell growth by a PHS-2 inhibitor, NS398, and a prostaglandin E receptor subtype EP1-selective antagonist, SC51089[END_REF].

Antitumor activity was evaluated by the change of relative tumor growth (RTG). RTG= (tumor volume on the 6 th daytumor volume before treatment)/tumor volume before treatment.

Measurement of DPD activity

The DPD enzyme activity of the cells and the xenografts was determined by radioenzymatic method as described previously [START_REF] Réti | Enhancement of 5-Fluorouracil Efficacy on High COX-2 Expressing HCA-7 Cells by Low-Dose Indomethacin and NS-398 but not on Low COX-2 Expressing HT-29 Cells[END_REF][START_REF] Reti | Co-inhibition of Cyclooxygenase-2 and Dihydropyrimidine Dehydrogenase by Non-steroidal Antiinflammatory Drugs in Tumor Cells and Xenografts[END_REF]. Measurements were made in parallel in three independent experiments.

Analysis of drug interaction

The effect of the combination of 5-FU and indomethacin or NS-398 was evaluated according to the method of Kern et al. [START_REF] Kern | In vitro pharmacodynamics of 1beta-D-arabinofuranosylcytosine: synergy of antitumor activity with cis-diamminedichloroplatinum(II)[END_REF]. The expected cell survival,(S exp ), defined as the product of the survival observed for 5-FU alone and the survival observed for indomethacin or NS-398 alone: S exp = (S 5-FU ) x (S indomethacin or NS-398 ) and the observed cell survival, (S obs ), obtained after combination of 5-FU and indomethacin or NS-398 were used to construct a synergistic ratio R = S exp / S obs (R) defining the drug interaction.. The values are: R = 1 additive effect; >1, synergistic effect; <1, antagonistic effect.

In case of xenografts the percentage ratio between the tumor volume of treated mice and the tumor volume of controls was used for the Kern's index calculation as described above.

Statistical analysis

Statistical analyses were performed using GraphPad PRISM software (San Diego, CA, USA).

The significance level was determined by analysis of variance (ANOVA) and Tukey's post hoc test. Differences between the collagen-induced and control cells were tested by using Student's t test for paired variants. The p values <0.05 were considered as significant.

Results

Expression of COX-2 and DPD in HT-29 cells induced by collagen

In order to verify if the ECM components may induce the COX-2 and DPD expressions, the HT-29 cells were cultured in the presence of collagen IV, fibronectin or laminin. The 4-ho ur incubation of HT-29 cells on collagen IV, caused significant increase of COX-2 and DPD protein expressions, (Fig. 1) but the fibronectin and laminin treatments were ineffective (data not shown). At the same time the PGE 2 level and DPD activities were significantly upregulated as well. DPYD and COX-2 mRNA expressions also showed a 287 and 750 fold increase, respectively, compared to the control cells (Table 1).

Influence of indomethacin or NS-398 on 5-FU cytotoxicity in HT-29, HT-29-C cells

In the uninduced HT-29 cells after 8-hour treatment 5-FU alone caused a very moderate cell growth inhibition which could not be potentiated with NSAIDs.

In HT-29-C cells 5-FU was almost ineffective in growth inhibition, however, the combination of 5-FU with indomethacin or NS-398 resulted in a remarkable growth inhibition (37% and 41% inhibition vs control, respectively) (Fig 2 .). The potentiation of 5-FU action by NSAIDs was similar to our earlier results obtained in HCA-7 cells, which demonstrated that indomethacin or NS-398 significantly enhanced the 5-FU cytotoxicity after 48-hour treatment ( 35% and 38% vs. 5-FU, respectively) [START_REF] Réti | Enhancement of 5-Fluorouracil Efficacy on High COX-2 Expressing HCA-7 Cells by Low-Dose Indomethacin and NS-398 but not on Low COX-2 Expressing HT-29 Cells[END_REF]. It is noteworthy, however, that in HT-29-C cells, characterized by enhanced COX-2 and DPD expressions, the potentiation of 5-FU action could be achieved already after 8-hour treatment compared to the constitutively high COX-2 and DPD expressing HCA-7 cells where 48 hours were necessary to obtain a significant 5-FU cytotoxic effect.

Influence of indomethacin or NS-398 on DPD mRNA expression and enzyme activity in HT-

29-C and HCA-7 cells

We have examined whether treatment of HT-29-C and HCA-7 cells with 5-FU and its combination with NSAIDs regulates the DPYD mRNA expression and enzyme activity one of the most important determinant of 5-FU action. In HT-29-C cells the DPYD mRNA expression was significantly decreased by 5-FU treatment (p< 0.01). This effect was potentiated by combining 5-FU with NSAIDs (p<0.05 vs 5-FU).

In case of HCA-7 cells, however, the DPYD mRNA expression was reduced in a nonsignificant manner, but when 5-FU was combined with indomethacin or NS-398 the decrease was significant compared to 5-FU treatment (p<0.05 vs 5-FU). No differences between the enhancing effect of indomethacin and NS-398 could be found (Table 2). The decrease of DPD enzyme activity was non-significant in both cell lines after 5-FU treatment, while at the same time indomethacin or NS-398 potentiated significantly the enzyme inhibitory effect of 5-FU.

(Table . 2)

Influence of indomethacin or NS-398 on the tumor growth inhibitory effect of 5-FU in HT-29 xenografts

The mice tolerated well the administration of 6 and 30 mg/kg/day 5-FU or their combinations with 2.5 mg/kg/day indomethacin or 5 mg/kg/day NS-398, as well.

In HT-29 xenografts 6 mg/kg 5-FU did not affect the tumor growth compared to the control, but the simultaneous treatment with indomethacin or NS-398 potentiated significantly the tumor growth inhibitory effect of 5-FU (33.3% and 42% decrease vs 5-FU). The tumor growth inhibitory potential of 6 mg/kg 5-FU + NSAIDs was similar to that obtained by 30 mg/kg 5-FU monotherapy, which decreased the relative tumor growth by ~50% (p<0.001) compared to the control, but it is noteworthy that NSAIDs could not further enhance the cytotoxic effect of 30 mg/kg 5-FU (Fig. 3). Neither indomethacin, nor NS-398 alone influenced significantly the tumor growth of HT-29 xenografts after 5-day treatment (data not shown).

Effect of 5-FU ± NSAIDs on DPD enzyme activity in HT-29 xenografts

5-fluorouracil (6 mg/kg/day) caused a significant decrease of DPD enzyme activity compared to the control and this enzyme inhibitory effect was potentiated significantly by INDO or NS-398 (p<0.05 and p<0.01, respectively) compared to 5-FU treatment. Noteworthy, that the decrease in DPD activity caused by the 6 mg/kg 5-FU+NSAIDs was similar to that obtained after treatment with 30 mg/kg 5-FU alone (Fig. 4). Similarly to the in vitro results the DPD suppressing effect of indomethacin or NS-398 was about the same.

Evaluation of drug interaction between 5-FU and NSAIDs

To investigate the therapeutic advantage of the potentiation of 5-FU cytotoxicity by NSAIDs the drug interaction analysis was performed. The type of interaction between 5-FU and indomethacin or NS-398 in HT-29-C cells was synergistic (R:1.57 and 1.71, respectively) compared to HT-29 cells in which it was only additive. A remarkable synergy was achieved also in case of HT-29 xenografts after treatment with 6mg/kg 5-FU combined with the two NSAIDs (R:1.48 and 1.47, respectively) (Table 3).

Discussion

Cyclooxygenase-2 expression was described in several tumor types including colorectal, gastric, esophageal, hepatocellular, breast etc. cancers and its overexpression is often associated with a shorter time to progression or overall survival [START_REF] De Groot | Non-steroidal anti-inflammatory drugs to potentiate chemotherapy effects: from lab to clinic[END_REF]. The cyclooxygenase enzymes are the best characterized targets of NSAIDs, which inhibit their activity. Recently, there has been much interest in using COX-2 inhibitors along with conventional anticancer therapy [START_REF] Sweeney | Why cyclooxygenase-2 inhibition plus chemotherapy?[END_REF]. Previously, it was demonstrated that in high COX-2 expressing cell lines NSAIDs in addition to COX-2 inhibition also reduced the DPD enzyme activity [START_REF] Reti | Co-inhibition of Cyclooxygenase-2 and Dihydropyrimidine Dehydrogenase by Non-steroidal Antiinflammatory Drugs in Tumor Cells and Xenografts[END_REF].

In the present study high COX-2-expressing colorectal cancer cell lines and xenograft were used to demonstrate the enhancement of 5-FU cytotoxicity induced by the NSAIDs-related inhibition of DPD mRNA level and enzyme activity.

The results of the present study regarding the induction of COX-2 by collagen IV are in accordance with the findings demonstrated that various ECM proteins induced COX-2 expression in HUVEC cells [START_REF] Zaric | Integrin-mediated adhesion and soluble ligand binding stabilize COX-2 protein levels in endothelial cells by inducing expression and preventing degradation[END_REF]. Collagen I and IV mediated expression of COX-2 was also described in epithelial cells [START_REF] Broom | Alpha2beta1 integrin signalling enhances cyclooxygenase-2 expression in intestinal epithelial cells[END_REF]. Until now, direct association between DPD expression and signals induced by ECM components could not be found in the literature. In 3D collagen I gel cultures the Sp1 transcription factor was increased in HSC cells [START_REF] Takahra | Induction of myofibroblast MMP-9 transcription in three-dimensional collagen I gel cultures: regulation by NF-kappaB, AP-1 and Sp1[END_REF]. Zhang et al.

demonstrated that the Sp1 binds to the DPYD promoter and acts as a potent transactivator [START_REF] Zhang | The role of Sp1 and Sp3 in the constitutive DPYD gene expression[END_REF]. Based on these data, one can speculate that collagen could affect the DPD expression via Sp1. On the other hand, NSAIDs activate proteosome-dependent degradation of Sp1 [START_REF] Abdelrahim | Cyclooxygenase-2 inhibitors decrease vascular endothelial growth factor expression in colon cancer cells by enhanced degradation of Sp1 and Sp4 proteins[END_REF].

In this study NSAIDs enhanced the cytotoxicity effect of 5-FU in vitro and in vivo, as well.

Many studies proved that NSAIDs potentiated the effect of conventional chemotherapeutic drugs such as 5-FU, cisplatin, oxaliplatin, S-1 etc [START_REF] Ogino | Indomethacin preferentially augments 5-fluorouracil cytotoxicity in Colon 26 tumors by increasing the intracellular inflow of 5-fluorouracil[END_REF][START_REF] Tachimori | Combination the rapy of S-1 with selective cyclooxygenase-2 inhibitor for liver metastasis of colorectal carcinoma[END_REF][START_REF] Leonetti | Efficacy of a nitric oxide-releasing nonsteroidal anti-inflammatory drug and cytotoxic drugs in human colon cancer cell lines in vitro and xenografts[END_REF]. However, there are only few investigations presenting the effect of 5-FU combined with low dose NSAIDs on cancer cells or xenografts. Low dose indomethacin significantly reduced the IC 50 of 5-FU treatment on COX-2-expressing murine Colon 26 cells [START_REF] Ogino | Indomethacin preferentially augments 5-fluorouracil cytotoxicity in Colon 26 tumors by increasing the intracellular inflow of 5-fluorouracil[END_REF]. Similarly, SKG-2 and HKUS human cervical cancer cells after treatment with indomethacin showed significantly increased 5-FU cytotoxicity [START_REF] Ogino | Indomethacin increases the cytotoxicity of cis-platinum and 5-fluorouracil in the human uterine cervical cancer cell lines SKG-2 and HKUS by increasing the intracellular uptake of the agents[END_REF]. An impressive but interestingly non-significant tumor volume decrease (40%) after 28-day low dose ibuprofen+5-FU treatment in a COX-2-expressing mouse colorectal tumor modell was described [START_REF] Yao | Effects of nonselective cyclooxygenase inhibition with low-dose ibuprofen on tumor growth, angiogenesis, metastasis, and survival in a mouse model of colorectal cancer[END_REF].

On the other hand, NS-398 at low dose protected HeLa cells against the cytotoxic action of cisplatin, paclitaxel and 5-FU [START_REF] Eichele | Decisive role of cyclooxygenase-2 and lipocalin-type prostaglandin D synthase in chemotherapeutics-induced apoptosis of human cervical carcinoma cells[END_REF]. It is worth mentioning, that Shestopal et al demonstrated that HeLa cells showed the lowest DPD mRNA level among five investigated cell lines [START_REF] Shestopal | Molecular cloning and characterization of the human dihydropyrimidine dehydrogenase promoter[END_REF].

The selective COX-2 inhibitor, JTE-522, synergistically enhanced the cytotoxic effect of 5-FU against COX-2 expressing bladder cancer cells, T24 and HT1197 [START_REF] Mizutani | Synergistic cytotoxicity and apoptosis of JTE-522, a selective cyclooxygenase-2 inhibitor, and 5-fluorouracil against bladder cancer[END_REF]. Triptolide, which among others showed COX-2-inhibitory effect too, at low concentration presented synergistic effect with 5-FU on the growth of HT-29 colon carcinoma in vitro and in vivo [START_REF] Tang | Synergistic effect of triptolide combined with 5-fluorouracil on colon carcinoma[END_REF].

Interestingly, in COX-2 expressing LM-H3 colon cancer cells the effect of 5-FU was enhanced by the DPD inhibitor, 5-chloro-2,4-dihydroxypyridine (CDHP). Etodolac and rofecoxib did not increase the cytotoxic effect of 5-FU, however, the combination of S-1 + NSAIDs inhibited the liver metastasis formation compared to S-1 after tumor cell inoculation in the mice spleen [START_REF] Tachimori | Combination the rapy of S-1 with selective cyclooxygenase-2 inhibitor for liver metastasis of colorectal carcinoma[END_REF]. Low dose of nitric oxide-donating NSAID, NCX 4040, enhanced 5-FU cytotoxicity on colon adenocarcinoma cell lines, but not xenografts [START_REF] Leonetti | Efficacy of a nitric oxide-releasing nonsteroidal anti-inflammatory drug and cytotoxic drugs in human colon cancer cell lines in vitro and xenografts[END_REF]. Their findings on cell lines are in accordance with the results of the present study, while the discrepant results regarding the xenografts may be due to the different treatment schedules and applied 5-FU doses.

Combination of 5-FU with various modulators e.g. DPD inhibitors, resulted in an increased antitumor effect and enhanced rate of apoptosis [START_REF] Ahmed | Eniluracil treatment completely inactivates dihydropyrimidine dehydrogenase in colorectal tumors[END_REF][START_REF] Kralovánszky | 5-Ethyl-2'-deoxyuridine, a modulator of both antitumour action and pharmacokinetics of 5-fluorouracil[END_REF][START_REF] Adeyemo | Antioxidants enhance the susceptibility of colon carcinoma cells to 5-fluorouracil by augmenting the induction of the bax protein[END_REF].

The present study is one of the first, which demonstrates that the NSAIDs caused decrease of DPD activity caused by NSAIDs enhanced the 5-FU cytotoxicity. Although, the DPD enzyme activity in connection with 5-FU action has been in limelight, up to this day there are few data about the DPD regulation, although, growing evidence has been accumulated that not only the well-known DPD inhibitors are able to modulate the DPD enzyme activity e.g TRAIL, IFN [START_REF] Mizutani | Potentiation of the sensitivity of renal cell carcinoma cells to TRAIL-mediated apoptosis by subtoxic concentrations of 5-fluorouracil[END_REF][START_REF] Dou | Consensus interferon enhances the anti-proliferative effect of 5-fluorouracil on human hepatoma cells via downregulation of dihydropyrimidine dehydrogenase expression[END_REF]. A very recent article described synergistic effect of 5-FU and a bioactive flavonoid, oroxylin A in vitro and in vivo. The combined treatment significantly decreased the DPYD mRNA levels compared to 5-FU alone. In addition, oroxylin A among other effects inhibits the lipopolysaccharide-induced COX-2 expression [START_REF] Zhao | Synergistic effect of 5-fluorouracil and flavonoid oroxylin A on HepG2 human hepatocellular carcinoma and on H 22 transplanted mice[END_REF].

DPD activity has previously been reported to correlate with its prote in level in human lymphocytes [START_REF] Takechi | Relationship between protein levels and gene expression of dihydropyrimidine dehydrogenase in human tumor cells during growth in culture and in nude mice[END_REF] and with mRNA levels in colorectal tumors and liver tissues [START_REF] Johnson | Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction[END_REF]. In the present study a similar correlation was found in vitro between DPD enzyme activity and the mRNA expression. On the other hand, others did not find a ny correlation between DPD enzyme activity, protein level and mRNA expression [START_REF] Miyamoto | Discrepancies between the gene expression, protein expression, and enzymatic activity of thymidylate synthase and dihydropyrimidine dehydrogenase in human gastrointestinal cancers and adjacent normal mucosa[END_REF]. The discrepancy might be due to the non-comparable methods used in different studies.

It is worth mentioning that in our study 5-FU alone caused a decrease in DPD enzyme activity both in vitro and in vivo. Literature data concerning the effect of 5-FU exposure on DPD levels are not conclusive [START_REF] Nishiyama | Low-dose cisplatin and 5-fluorouracil in combination can repress increased gene expression of cellular resistance determinants to themselves[END_REF][START_REF] Sakurai | Changes in thymidylate synthase and its inhibition rate and changes in dihydropyrimidine dehydrogenase after the administration of 5-fluorouracil with cisplatin to nude mice with gastric cancer xenograft SC-1-NU[END_REF][START_REF] Sakurai | Changes of gene expression of thymidine phosphorylase, thymidylate synthase, dihydropyrimidine dehydrogenase after the administration of 5'-deoxy-5-fluorouridine, paclitaxel and its combination in human gastric cancer xenografts[END_REF] thus further investigations are needed to clarify in details these findings.

Although, it is well known that 5-FU resistance is multifactorial the DPD status is among the most important predictors of 5-FU efficacy in vitro, in vivo and in clinical practice, as well.

The sensitivity of 4 gastric and 3 colorectal cancer cell lines to 5-FU were in inverse correlation with DPD protein or mRNA levels [START_REF] Ma | Correlation of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2'-deoxyuridine[END_REF]. A significant correlation was found between DPYD DNA copy number and mRNA or enzyme activity in 58 human cancer cell lines and 31 human cancer xenografts. In cell lines the 5-FU sensitivity was significantly correlated with DPYD copy number, which also differed significantly between drug-resistant and drug-sensitive xenografts [START_REF] Kobunai | Changes to the dihydropyrimidine dehydrogenase gene copy number[END_REF]. The prognostic value of DPD enzyme activity [42] or mRNA level [43] was demonstrated in patiens with colorectal cancer treated with 5-FU-based chemotherapy.

In the present study the indomethacin and NS-398 caused downregulation of DPYD mRNA expression and consequently the reduction of the enzyme activity in expreimental systems was in good relationship with the enhancement of the antiproliferative potency of 5-FU.

  indomethacin and NS-398 in parallel with the COX-2 inhibition decreased also the DPD enzyme activity of high COX-2-expressing HCA-7 cells and HT-29 xenografts. It is noteworthy that the HT-29 xenograft was characterized by high COX-2 and DPD enzyme activity after a phenotype change of the implanted low COX-2 and DPD expressing HT-29 cells [9]. To elucidate the possible mechanism of this phenotype change in the present study the HT-29 cells were cultured with extracellular matrix (ECM) components i.e. fibronectin, laminin or collagen IV in order to see if they are able to modify the COX-2 and DPD expression of the cells. Moreover, it was intended to prove whether the 5-FU cytotoxicity could be increased by combination with NSAIDs in the collagen IV-induced HT-29 or in the constitutively high COX-2-expressing HCA-7 cells and in the HT-29 xenografts through the modulation of DPD mRNA expression and/or enzyme activity.

Fig 1 Fig 2 FUFig 3

 123 Fig 1 Representative immunofluorescent staining of COX-2 in HT-29 (a) and collagen-

(a) 6 Fig 4

 64 Fig 4 Inhibitory effect 5-FU (open bar) 5-FU + indomethacin (gray bar) and 5-FU+NS-398

Table 1 .

 1 Comparison of cyclooxygenase-2 and dihydropyrimidine dehydrogenase status of

	uninduced HT-29 and collagen-induced HT-29-C cells
		HT-29	HT-29-C
	DPD enzyme activity (pmol/min/mg	9 ± 2.7	17.44 ± 2.87 #
	protein)
	PGE 2 level (pg/ml)	ND	50.85 ± 12.3 #
	Relative DPYD mRNA expression a	8.09 ± 0.4 x 10 -3 2328 ± 464x10 -3 #
	Relative COX-2 mRNA expression a	4.8 ± 1.4 x 10 -6	3624 ± 1221x10 -6
			#
	a related to ß-actin mRNA; # p<0.05 vs. uninduced cells; ND: non-detectable;

Table 2 .

 2 Change of dihydropyrimidine dehydrogenase status after the treatment with 5-FU ±

	indomethacin or NS-398 in collagen-induced HT-29 (HT-29-C) and HCA-7 cells
		HT-29-C cells	HCA-7 cells
		mRNA	enzyme activity b	mRNA	enzyme activity b
		expression a		expression a
	control	2.3 ± 0.5	17.4 ± 2.9	10.4 ± 3.7	99.6 ± 13.1
	5-FU	0.9 ± 0.08 *	14.6 ± 3.6	9.6 ± 1.4	90.2 ± 1.7
	5-FU+	0.06 ± 0.04 #	5.1 ± 1.6 #	1.2 ± 0.4 #	54.3 ± 7.6 #
	indomethacin
	5-FU+ NS-398	0.005 ± 0.004 #	5.9 ± 1.1 #	0.6 ± 0.07 #	55.0 ± 6.3 #
	HT-29 cells were incubated with 10 M 5-FU alone or combined with either 10 M
	indomethacin or 1.77 M NS-398; HCA-7 cells were incubated with 1mM 5-FU alone or
	combined with either 10 M indomethacin or 1.77 M NS-398: a related to ß-actin mRNA; b
	pmol/min/mg protein, * p< 0.01 vs control; # p<0.05 vs 5-FU

Table 3 .

 3 Drug interaction between 5-FU and indomethacin or NS-398

		R INDO	a	R NS-398	a
	HT-29 cells	1.02		1.09
	HT-29-C cells	1.57		1.71
	HT-29 xenograft 6 mg/kg 5-	1.48		1.47
	FU
	HT-29 xenograft 30 mg/kg 5-	1.19		1.13
	FU

a Type of interaction R= 1, additive effect; >1, synergistic effect; <1, antagonistic effect. The synergistic ratio R, was calculated as: R = S exp /S obs

Finally, based on our results it is possible to conclude that 5-FU efficacy could be limited by the COX-2 associated high DPD expression and activity in patients with colorectal cancers as well, therefore further clinical studies are varraned to decide if NSAIDs in the therapeutic protocol might inprove the antitumor potency of 5-FU.

Acknowledge ments

The SCID mice were generously gifted by Dr. D. Gaál. The excellent technical assistance of Cs. Polényi Makácsné, A. Nagy, J. Kútvölgyi, A. Éber Mousáné, A. Sztodola and A. M. Borza were greatly appreciated.

This study was supported by the Jedlik Ányos Grant (NKFP1-00024/2005).