Synthetic and naturally occurring COX-2 inhibitors suppress proliferation in a human oesophageal adenocarcinoma cell line (OE33) by inducing apoptosis and cell cycle arrest

E. Cheong1,2, K. Ivory2, J. Doleman2, M.L. Parker2, M. Rhodes1 and I.T. Johnson2,3

1 General Surgery Department, Norfolk and Norwich University Hospital, Colney, Norwich NR4 7UY, UK and 2 Nutrition and Gastrointestinal Health Programme, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

3 To whom correspondence should be addressed. Tel: +44 1603 255330; Fax: +44 (0) 1603 255167; Email: ian.johnson{at}bbsrc.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies suggest that the use of NSAIDs and/or a high intake of fruit and vegetables reduce the risk of oesophageal adenocarcinoma. Since COX-2 is up-regulated in Barrett's oesophageal carcinogenesis, the protective effect of NSAIDs and natural food components might reflect COX-2 inhibition. We explored the effects of quercetin, a natural flavonoid with a potent COX-2 inhibitory activity, and two commercially available selective COX-2 inhibitors (NS-398 and nimesulide) on cell proliferation, apoptosis, PGE2 production and COX-2 mRNA expression in a human oesophageal adenocarcinoma cell line (OE33). Changes in the relative numbers of adherent and floating cells were quantified and apoptotic cells were identified using ethidium bromide and acridine orange staining under fluorescence microscopy. Flow cytometric analysis of adherent and floating cells was used to quantify apoptosis and to examine the effects of the agents on the cell cycle. After 48 h exposure at concentrations of ≥1 µM both COX-2 inhibitors and quercetin suppressed cell proliferation (P < 0.01) and increased the fraction of floating apoptotic cells. At higher concentrations (50 µM) and longer exposure (48 h) the effects of quercetin were significantly greater than those of the selective COX-2 inhibitors (P < 0.01). Cell cycle analyses showed that quercetin blocked cells in S phase, while the selective COX-2 inhibitors blocked cells in G1/S interphase. COX-2 mRNA expression was suppressed by quercetin and the synthetic COX-2 inhibitors in a time- and dose-dependent manner. Quercetin and the synthetic COX-2 inhibitors (10 µM) suppressed PGE2 production by ~70% after 24 h exposure (P < 0.001). We conclude that OE33 is a useful model for the study of COX-2 expression and associated phenomena in human adenocarcinoma cells. Synthetic COX-2 inhibitors and the food-borne flavonoid quercetin suppress proliferation, induce apoptosis and cell cycle block in human oesophageal adenocarcinoma cells in vitro, and future studies should assess their effects in vivo.

Abbreviations: COX, cyclooxygenase; NSAIDs, non-steroidal anti-inflammatory drugs; NF-{kappa}B, nuclear factor {kappa}B; OAC, oesophageal adenocarcinoma; PBS, phosphate-buffered saline; PTKs, protein-tyrosine kinases; TGF{alpha}, transforming growth factor {alpha}


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of Barrett's oesophageal adenocarcinoma (OAC) is increasing rapidly in many Western countries (1,2). The reasons for this are unknown, but are believed to be linked to an increasing prevalence of the premalignant lesion Barrett's metaplasia (3). With an incidence of 16 per 100 000, compared with 5 per 100 000 in the USA, the UK has the highest incidence of OAC in the world (46). Despite advances in multimodality therapy for OAC, the overall 5 year survival rate has remained at <20% in the last 30 years (7).

There is a substantial body of epidemiological data supporting the role of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) in the chemoprevention of oesophageal cancer (8). The mechanism underlying this effect is not clearly established but is believed to involve inhibition of the enzyme cyclooxygenase (COX), which catalyses the rate limiting step in the biosynthesis of prostaglandins from arachidonic acid. There are two known isoenzymes of cyclooxygenase, COX-1 and COX-2; the former is constitutively expressed in most tissues, whereas COX-2 is induced by pro-inflammatory or mitogenic stimuli, including cytokines, growth factors and tumour promoters (9,10). Human and animal studies have indicated an important role for COX-2 in gastrointestinal carcinogenesis (1113). Overexpression of COX-2, and a concomitant overproduction of prostanoids, inhibits apoptosis and immune surveillance and increases cancer cell proliferation, tumour angiogenesis and invasiveness of malignant cells, thereby favouring malignant growth (14,15). Compared with normal squamous oesophageal epithelium, Barrett's oesophageal metaplasia, dysplasia and adenocarcinomas show overexpression of COX-2, but not COX-1 (1618).

Quercetin, a food-borne COX-2 inhibitor, belongs to an extensive class of polyphenolic flavonoid compounds that occur commonly in plants used as human food and in beverages, including tea and wine (19). Epidemiological studies suggest an inverse relationship between fruit and vegetable consumption and the risk of oesophageal adenocarcinoma (20), and a substantial reduction in risk of oesophageal cancer associated with intake of green tea (21,22). Quercetin is a potent inhibitor of COX-2 transcriptional activity (23) and a powerful antioxidant (24). In the present study we describe the use of an oesophageal adenocarcinoma cell line that expresses COX-2 (OE33) as a model system to explore the effects of COX-2 inhibitors on prostaglandin synthesis and cell growth and survival in oesophageal tissue. The system has been applied to quercetin as a prototype food-borne COX-2 inhibitor and to two synthetic selective COX-2 isoenzyme inhibitors, NS-398 and nimesulide.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell line and culture conditions
The OE33 cell line (passage 7), also known as JROECL33, was purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK). The cell line was established from a poorly differentiated lower oesophageal adenocarcinoma of a female Caucasian patient who had Barrett's metaplasia (25). The cells (passages 7–17) were cultured in RPMI 1640 medium (R0883; Sigma, Poole, UK) supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin G and 100 µg/ml streptomycin (all obtained from Imperial Laboratories, Andover, UK) and maintained in monolayer culture at 37°C in an incubator of humidified air with 5% CO2. Subconfluent cells (80%) were passaged with a solution containing 0.05% trypsin and 0.5 mM EDTA. The cells were plated for 24 h to allow them to attach, before treatment with quercetin (Q0125; Sigma) or the synthetic COX-2 inhibitors NS-398 and nimesulide (both from Cayman Chemical, MI). Each compound was dissolved in DMSO and made up with the medium so that the final concentration of the vehicle was not >0.5% DMSO. The cells were treated with the respective compounds at 0, 1, 10 and 50 µM, a concentration range that selectively inhibited the COX-2 isoenzyme with the synthetic COX-2 inhibitors used and also encompassed the concentration (IC50 = 10.5 µM) at which quercetin has previously been shown to inhibit COX-2 transcription (26). Cells treated with 0.5% DMSO served as a negative control.

Western blotting for COX-2
Cells were washed in phosphate-buffered saline (PBS), centrifuged to remove the supernatant and resuspended in 300 µl PBS, containing 75 µl mini-Complete Protease Inhibitor (Roche, Mannheim, Germany) and 125 µl NuPage LDS sample buffer (NP0007; Invitrogen, UK). The solution was triturated until it homogenized and kept on ice for 20 min. The cell suspension was probe-sonicated with three successive bursts for 10 s and subsequently incubated at 90°C for 10 min in a thermal mixer (Thermomixer 5436; Eppendorf). The sample was then centrifuged to sediment cellular debris. The protein supernatant was separated on 10% NuPage Bis–Tris gels (NP0302; Invitrogen, UK). COX-2 (ovine) electrophoresis standard (360120; Cayman Chemical) was used as a positive control. A recombinant protein molecular weight marker (Rainbow marker RPN 800; Amersham, UK) was also loaded. The proteins were transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, UK) using a semi-wet blotting method (XCell II Blot Module; Invitrogen, UK). The membrane was incubated in PBS/5% milk solution overnight at 4°C on a rotary shaker and then washed in PBS/0.1% Tween-20 (Sigma, UK) before it was probed with COX-2 monoclonal antibody (160112; Cayman Chemical) at 1:1000 dilution. A horseradish peroxidase-linked anti-mouse secondary antibody (NA931; Amersham Life Science, UK) was used at 1:5000 dilution. The protein bands were detected using an enhanced chemiluminescence procedure (ECL-PLUS detection kit; Amersham Pharmacia Biotech, UK).

COX-2 mRNA quantitation
OE33 cells (4 x 106 cells/flask) were plated in 75 cm2 flasks and left for 24 h to allow them to attach. The cells were treated with COX-2 inhibitors at 0, 1, 10 or 50 µM for 24 and 48 h. Colorimetric mRNA quantitation (RN000 and RN 171 Quantikine mRNA; R&D Systems, MN) was used to determine COX-2 mRNA level in 2 x 106 adherent cells according to the manufacturer's protocol. The 96-well plate was read in an automated microplate reader (7520 SpectraCount; Packard) set at 450 nm, with the reference filter set at 620 nm, and the results were calculated using I-Smart 2.0 (Packard). The experiment was repeated six times and the mean value was taken as the result.

PGE2 assay
OE33 cells (1 x 106 cells/flask) were plated in 25 cm2 flasks and left for 24 h to allow them to attach. Each flask was then treated with the respective COX-2 inhibitors at 0, 1, 10 and 50 µM so that the total volume of culture medium in each flask was only 5 ml. After 24 h, an aliquot of the culture medium (1 ml) was removed from each flask and any particulate was removed by centrifugation. PGE2 levels from the supernatants of the cell cultures were measured using a PGE2 competitive enzyme immunoassay (514010; Cayman Chemical) according to the manufacturer's protocol. Each PGE2 value was normalized for the number of cells attached. The 96-well plate was read in an automated microplate reader at 405 nm optical density and the results were calculated in I-Smart 2.0 (Packard). The experiment was performed in triplicate and the mean PGE2 value was taken as the result.

Cell viability assay
OE33 cells (15 000 cells in 200 µl medium/well) were plated in 96-well plates and incubated for 24 h to allow the cells to attach. They were then treated with each compound for 24, 48 and 72 h at 0, 1, 10 and 50 µM. Some cells were grown in 0.5% DMSO as a negative control. After each time point, the medium was removed and the cells were stained with neutral red solution for 2 h in the incubator. Subsequently the excess neutral red solution was removed and the cells were fixed in a solution of 0.5% formaldehyde and 0.1% calcium chloride. The plates were then washed twice with PBS solution and air dried. A solution of 50% ethanol and 1% acetic acid (200 µl) was delivered to each well and the plate was kept at 4°C overnight to allow complete dissolution of the dye. Each plate was read in an automated microplate reader at 550 nm optical density and the results were calculated in I-Smart 2.0 (Packard). Each experiment was repeated five times and the mean value was taken as the result.

Analysis for apoptosis
Since adherent cells tend to become detached early in apoptosis, the extent of apoptosis can be estimated from the proportion of floating cells in the total cell population (adherent and floating cells) of the culture (27). OE33 cells (2 x 106cells) were plated in 75 cm2 flasks for 24 h and then treated with quercetin or synthetic COX-2 inhibitors at 0, 10 and 50 µM for 48 h. Floating and adherent cells were collected separately and counted with a haemocytometer. To confirm that the floaters consisted of predominantly apoptotic cells, they were examined for morphological features of apoptosis under a fluorescence microscope after staining with a solution of acridine orange and ethidium bromide (each 5 µg/ml in PBS). Cells were identified as apoptotic on the basis of specific morphological criteria (28), including condensation and fragmentation of chromatin. The proportion of floating cells demonstrating apoptotic morphological features was determined by counting 200 cells in a random field at 40x magnification. Each experiment was performed in triplicate and the mean value was taken as the result.

Cell cycle analysis by flow cytometry
OE33 cells (5 x 105 cells) were plated in 25 cm2 flasks with 10 ml medium for 24 h. The cells were then treated for a further 48 h with quercetin or synthetic COX-2 inhibitors at 0, 10 or 50 µM. The floating and adherent cells were combined together for the analysis. Cells were washed in 5 ml of PBS and centrifuged at 800 r.p.m., before the supernatant was removed and the cells resuspended in 100 µl of PBS. The resuspended cells were stained according to the protocol for the Coulter DNA-Prep Reagents Kit (PN 6607055; Beckman Coulter, UK). DNA-prep LPR (Lyse) (100 µl) was added to the tube and the cell suspension was vortexed for 7 s, and then 1 ml DNA-prep stain (propidium iodide + RNase) (1 ml) was added. While staining, cells were placed in a darkened area for 30 min at room temperature prior to linear data acquisition on a Coulter EPICS Altra flow cytometer equipped with a water-cooled 488 nm Coherent 90 laser. Doublet discrimination was applied by plotting propidium iodide peak (y-axis) versus integral (x-axis) signals. A gate comprising a diagonal parallelogram was drawn around the event clusters on the assumption that aggregates should fall below the gated area as their pulse peak value would be lower than non-aggregates for a given integral signal. A minimum of 50 000 events were collected per sample. Acquired data were analysed using Expo2 (Beckman Coulter) and Multicycle (Phoenix Flow Systems, San Diego, CA) software.

COX inhibitor screening assay
To determine the ability of quercetin to inhibit the COX-1 and COX-2 isoenzymes, a COX inhibitor screening assay (560131; Cayman Chemical) was used according to the manufacturer's protocol. Resveratrol at 30 µM (Cayman Chemical) and NS-398 at 10 µM were used as positive controls for COX-1 and COX-2 isoenzyme inhibition, respectively. COX-1 and COX-2 isoenzymes treated with vehicle only (0.5% DMSO) served as the negative controls (100% activity). The concentrations of quercetin tested were 1, 10, 50, 100 and 1000 µM. The 96-well plate was read in an automated microplate reader (7520 SpectraCount; Packard) at 405 nm optical density and the results were calculated using a computer program (I-Smart 2.0; Packard).

Statistical analysis
Comparisons between treatment and control means were performed using two-tailed unpaired Student's t-tests, with the Bonferroni correction for multiple comparisons applied to the analyses of the effect of COX-2 inhibitors on cell viability and mRNA expression. Data are shown as means ± SEM. P values ≤ 0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
COX-2 expression in the OE33 cell line
To establish the COX-2 status of the OE33 human oesophageal adenocarcinoma cell line, Western blot analyses of cell lysates and colorimetric COX-2 mRNA quantitation of OE33 cells were performed. The results confirmed the constitutive expression of COX-2 protein and mRNA by OE33 cells (Figure 1).



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Fig. 1. Expression of COX-2 mRNA by OE33 cells and its suppression by COX-2 inhibitors. Cells were treated with quercetin (Q), NS-398 (NS) and nimesulide (NIM) at increasing concentrations from 0 to 50 µM for 24 and 48 h. Cells treated with the vehicle only served as a negative control. In each case 2 x 106 adherent cells were used for COX-2 mRNA quantification. Quercetin and the synthetic COX-2 inhibitors suppressed OE33 cellular COX-2 mRNA expression in a dose- and time-dependent manner. Data are expressed as means ± standard errors (n = 6).*P < 0.001 compared with the appropriate control.

 
Effects of COX-2 inhibitors on COX-2 mRNA expression
The effects of quercetin and the synthetic COX-2 inhibitors (NS-398 and nimesulide) on COX-2 mRNA expression were compared in cells treated for 24 and 48 h with COX-2 inhibitors over the concentration range 0–50 µM (Figure 1). After 24 h treatment at 10 µM only quercetin significantly suppressed COX-2 mRNA expression when compared with the control (P < 0.001). However, when the concentrations of the synthetic COX-2 inhibitors were increased to 50 µM, COX-2 mRNA expression was also significantly suppressed compared with the control (P < 0.001). After 48 h treatment, further suppression of COX-2 mRNA expression was seen (P < 0.03). This was seen even at the lower concentrations of quercetin (1 µM) and synthetic COX-2 inhibitors (10 µM) used. Hence, both quercetin and the synthetic COX-2 inhibitors suppressed COX-2 mRNA expression in OE33 cells in a dose- and time-dependent manner.

Effects of COX-2 inhibitors on PGE2 production
The effects of quercetin and the synthetic selective COX-2 inhibitors on PGE2 production by OE33 cells were explored (Figure 2). After 24 h treatment, PGE2 production was significantly suppressed by both quercetin and the synthetic COX-2 inhibitors at 10 µM compared with the negative control (P < 0.001). No further significant suppression of PGE2 production was seen when the concentrations of the COX-2 inhibitors were increased to 50 µM.



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Fig. 2. PGE2 levels in the supernatant of OE33 cell cultures measured after 24 h treatment with COX-2 inhibitors. The values were normalized for the number of adherent cells. The negative control was treated with the vehicle (0.5% DMSO) only. Quercetin (Q) and the synthetic COX-2 inhibitors NS-398 (NS) and nimesulide (NIM) significantly suppressed PGE2 production compared with the control. No further significant reduction in PGE2 level was seen when the doses were increased from 10 to 50 µM. Data are expressed as means ± standard errors (n = 3). *P < 0.001.

 
Effects of COX-2 inhibitors on OE33 cell growth
The OE33 cells were treated with quercetin or the synthetic COX-2 inhibitors at concentrations of 1, 10 and 50 µM for 24, 48 and 72 h (Figure 3). Compared with the negative control, both quercetin and the synthetic COX-2 inhibitors significantly suppressed OE33 cell growth in a dose- and time-dependent fashion (P < 0.01). However, quercetin had a significantly greater inhibitory effect on OE33 cell proliferation than the synthetic COX-2 inhibitors (P < 0.01).



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Fig. 3. Inhibition of OE33 cell growth by COX-2 inhibitors. Cells were treated with the COX-2 inhibitors at 1, 10 and 50 µM over 24, 48 and 72 h. The results are shown as percentages of the negative control, which was treated with the vehicle only. Quercetin and the synthetic COX-2 inhibitors significantly suppressed OE33 cell growth in a dose- and time-dependent manner. Data are expressed as means ± standard errors (n = 5). *P < 0.001 compared with the appropriate control.

 
Effects of COX-2 inhibitors on apoptosis
The majority of floating cells occurring spontaneously in a culture have been shown to be apoptotic (29). To determine whether the growth inhibitory effects of the COX-2 inhibitors on OE33 cells were associated with an increase in the proportion of floating cell in our system, the total cell population of the culture was counted (Figure 4). There was a significantly higher proportion of floaters after OE33 cells were treated with quercetin (P < 0.001) or the synthetic COX-2 inhibitors (P < 0.001) when compared with the negative control. To confirm the induction of apoptosis, the floaters were examined under a fluorescence microscope after staining with acridine orange and ethidium bromide solution. Apoptotic cells were bright green or orange cells with condensed and/or fragmented nuclear chromatin (Figure 5). The majority (90%) of floaters were found to be apoptotic cells, whereas adherent cells did not display an apoptotic morphology to any significant extent. It was inferred therefore that quercetin and the synthetic COX-2 inhibitors inhibit OE33 cell growth primarily by inducing apoptosis.



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Fig. 4. Induction of apoptosis in OE33 cells by COX-2 inhibitors. The majority of spontaneously occurring floating cells in a culture have been shown to consist of apoptotic cells. The extent of apoptosis was shown by measuring the proportion of floating cells in the total cell population (adherent and floating cells) of the culture. Results are shown as multiples with respect to the control at 48 h after treatment. Data are expressed as means ± standard errors. Quercetin and the synthetic COX-2 inhibitors significantly induced apoptosis in OE33 cells (n = 3). *P < 0.001 compared with the control.

 


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Fig. 5. Floating OE33 cells taken from a culture treated for 48 h with quercetin (50 µM), showing features of apoptosis (condensation and fragmentation of nuclear chromatin). Cells were stained with acridine orange and ethidium bromide and examined by fluorescence microscopy. Scale bar = 10 µm.

 
Effect of COX-2 inhibitors on OE33 cell cycle
Flow cytometry was performed to determine the effects of the synthetic COX-2 inhibitors and quercetin on cell cycling. The synthetic COX-2 inhibitors suppressed cell growth by blocking the cell cycle predominantly at G1/S interphase, while quercetin blocked the cell cycle predominantly in S phase (Figure 6). Cells treated with synthetic COX-2 inhibitors had a significant sub G1 peak representing the apoptotic cells. Quercetin-treated cells had a significantly higher growth inhibition and apoptosis. When the analyses included only adherent cells, no apoptotic cells were detectable (data not shown).



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Fig. 6. Cell cycle analyses after 48 h treatment with either the synthetic COX-2 inhibitor nimesulide or quercetin at 50 µM concentration, compared with the control. The synthetic COX-2 inhibitors and quercetin blocked OE33 cell cycling at the G1/S interphase and S phase, respectively.

 
COX inhibitor assay
Assays were performed to determine the inhibitory activity of quercetin against the COX-1 and COX-2 isoenzymes. Resveratrol (30 mM) and NS-398 (10 mM) were used as COX-1 and COX-2 positive controls, respectively. Quercetin at concentrations of up to 50 µM had no detectable COX-1 or COX-2 isoenzyme inhibitory activity, but at 100 and 1000 µM quercetin caused relatively weak inhibition of both the COX-1 and COX-2 isoenzymes (Figure 7). The positive controls, resveratrol and NS-398, significantly inhibited the COX-1 and COX-2 isoenzymes, respectively.



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Fig. 7. Inhibition of the COX-1 and COX-2 isoenzymes by quercetin. Resveratrol (30 µM) and NS-398 (10 µM) were used as positive controls for inhibition of the COX-1 and COX-2 isoenzymes, respectively. At concentrations <50 µM, quercetin did not have any inhibitory activity on the COX-1 and COX-2 isoenzymes. Only at concentrations of ≥100 µM did quercetin significantly inhibit both COX-1 and COX-2 isoenzymes activities. Data are expressed as means ± standard errors (n = 3). *P < 0.001 compared with the appropriate control.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is persuasive evidence that diets containing relatively large quantities of fruits and vegetables are associated with a reduced risk of oesophageal cancer (20,30). Such diets are likely to be rich in conventional nutrients, but also in biologically active secondary metabolites, amongst which the flavonoids display a spectrum of biochemical activities that may contribute to the chemoprevention of cancer. These effects include antiproliferative and anticarcinogenic properties (31), such as free radical scavenging, inhibition of lipid peroxidation (32) and suppression of COX-2 transcription (23). Increased COX-2 expression occurs in Barrett's oesophagus (17) and in Barrett's epithelial cells in response to bile acid and/or gastric acid, both in vitro (33) and in animal models (34). Conversely, suppression of COX-2 expression in Barrett's epithelial cells by selective COX-2 inhibitors is associated with inhibition of cell proliferation both in vitro (16) and in vivo (35). Thus natural, food-borne COX-2 inhibitors such as quercetin and other flavonoids (36) may play a role in the protective effects of fruit- and vegetable-rich diets against oesophageal adenocarcinoma.

In the present study we have shown that the Barrett's oesophageal adenocarcinoma cell line OE33 expresses both COX-2 mRNA and enzyme activity in the unstimulated state. Both the synthetic COX-2 inhibitors and quercetin suppressed COX-2 mRNA expression in a time- and dose-dependent manner, inhibited cell proliferation and simultaneously increased the fraction of floating apoptotic cells in the system. Indeed, the effects of quercetin were significantly greater than those of the selective COX-2 inhibitors. Quercetin also suppressed PGE2 production in OE33 cells independently of any direct inhibition of the COX-1 or COX-2 isoenzymes. Its ability to inhibit phospholipase A2 (37) and to down-regulate COX-2 expression could explain the suppression of PGE2 by OE33 cells. It should be noted that quercetin aglycone, the form used in the present study, is not normally present in human plasma because the aglycone is rapidly metabolized by the intestinal mucosa and subsequently by the liver (38). However, in oral administration studies the major circulating forms of quercetin present have been shown to be quercetin 3-glucuronide, 3'-methylquercetin 3-glucuronide and quercetin 3'-sulphate (38); all of these metabolites are now known to inhibit COX-2 expression in human lymphocytes in vitro (39).

Cell cycle analyses showed that while the selective COX-2 inhibitors blocked the cell in G1/S interphase, quercetin blocked the cell in S phase. Quercetin is a potent inhibitor of tyrosine kinase and protein kinase C, both of which are involved in COX-2 transcription and in cell proliferation and transformation (40–42). It is well established that transforming growth factor {alpha} (TGF{alpha}) and 12-O-tetradecanoylphorbol-13-acetate, both of which stimulate COX-2 expression in cancer cells (26), activate protein-tyrosine kinases (PTKs) (43). TGF{alpha} binds to epidermal growth factor receptor (a receptor PTK) and the activated PTKs transduce signals downstream via pathways involved in the induction of COX-2 gene expression. Hence, quercetin, like resveratrol, may inhibit COX-2 expression in OE33 cells by inhibiting the activation of PTKs (44).

It is increasingly recognized that the antiproliferative effects of selective COX-2 inhibitors against cancer cells can also occur via cyclooxygenase-independent mechanisms (45), and the same may be true of quercetin. For example, COX-2 inhibitors cause activation of nuclear factor {kappa}B (NF-{kappa}B) and NF-{kappa}B-dependent gene transcription (46) and sensitization to apoptosis-inducing treatments such as chemo- or radiotherapy (47). As another example, the selective COX-2 inhibitor celecoxib induces apoptosis in prostate cancer cells by reducing phosphorylation of the anti-apoptotic kinase Akt and, hence, blocking its anti-apoptotic activity (48).

Synthetic COX-2 inhibitors can also induce cell cycle arrest independently of COX-2 suppression (45), apparently by reducing expression of regulatory elements involved in cell cycle progression, notably cyclins and cyclin-dependent kinases, while increasing the expression of cell cycle arrest genes (49).

In conclusion, we have shown that the human adenocarcinoma cell line OE33 can be used as a model for the inhibition of COX-2 activity in vitro and that both selective COX-2 inhibitors and the natural flavonoid quercetin can suppress proliferation, induce apoptosis and cause cell cycle arrest in this cell line. OE33 is therefore a potentially valuable in vitro system for the evaluation of novel chemopreventive agents against oesophageal adenocarcinoma. Quercetin was shown to exert an even greater inhibitory effect than synthetic COX-2 inhibitors on these cells, but further studies will be required to determine whether quercetin and other naturally occurring COX-2 inhibitors might also exert anticarcinogenic effects against oesophageal adenocarcinoma in vivo.


    Acknowledgments
 
This work was partially funded by a core strategic grant of the BBSRC. I.T.J. acknowledges financial support from an EC funded programme on ‘Health implications of natural non-nutrient antioxidants’ (POLYBIND, QLK1 1999.00505).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 1, 2004; revised April 29, 2004; accepted May 1, 2004.