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Endocrinology Vol. 147, No. 9 4505-4516
Copyright © 2006 by The Endocrine Society

Leptin Stimulates Proliferation and Inhibits Apoptosis in Barrett’s Esophageal Adenocarcinoma Cells by Cyclooxygenase-2-Dependent, Prostaglandin-E2-Mediated Transactivation of the Epidermal Growth Factor Receptor and c-Jun NH2-Terminal Kinase Activation

Olorunseun Ogunwobi, Gabriel Mutungi and Ian L. P. Beales

Biomedical Research Centre (O.O., G.M., I.L.P.B.), School of Medicine, Health Policy, and Practice, University of East Anglia, Norwich NR4 7TJ, United Kingdom; and Department of Gastroenterology (I.L.P.B.), Norfolk and Norwich University Hospital, Norwich NR4 7UZ, United Kingdom

Address all correspondence and requests for reprints to: Dr. Ian Beales, School of Medicine, Health Policy, and Practice, University of East Anglia, Norwich NR4 7TJ, United Kingdom. E-mail: i.beales{at}uea.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Obesity is an important risk factor for esophageal adenocarcinoma (EAC), and elevated serum leptin is characteristic of obesity. We hypothesized that leptin may have biological effects in promoting esophageal adenocarcinoma and examined the effects of leptin on the OE33 Barrett’s-derived EAC line. Proliferation was assessed by dimethylthiazoldiphenyltetra-zoliumbromide and 5-bromo-2'-deoxyuridine incorporation assays and apoptosis by ELISA of intracellular nucleosomes. Intracellular signaling was examined using specific pharmacological inhibitors and direct detection of phosphorylated active kinases. Expression of the long and short leptin receptors by OE33 cells was confirmed by RT-PCR, Western blotting and immunocytochemistry. Leptin stimulated OE33 cell proliferation in a dose-dependent manner and inhibited apoptosis. These effects were dependent on cyclooxygenase (COX)-2 and replicated by adding prostaglandin E2 (PGE2). The effects of PGE2 and leptin were abolished by the EP-4 antagonist AH23848. ERK, p38 MAPK, phosphatidylinositol 3'-kinase/Akt, and Janus tyrosine kinase (JAK)-2 were activated upstream of COX-2 induction, whereas the epidermal growth factor receptor and c-Jun NH2-terminal kinase (JNK) were downstream of COX-2. The activation of ERK and Akt but not p38 MAPK was JAK2 dependent. PGE2 stimulated phosphorylation of JNK in an EGF receptor-dependent manner, and activation of the epidermal growth factor receptor required protein kinase C, src, and matrix metalloproteinase activities. We conclude that leptin stimulates cell proliferation and inhibits apoptosis in OAC cells via ERK, p38 MAPK, phosphatidylinositol 3'-kinase/Akt, and JAK2-dependent activation of COX-2 and PGE2 production. Subsequent PGE2-mediated transactivation of the epidermal growth factor receptor and JNK activation are essential to the leptin effects. These effects may contribute to the greatly increased risk of esophageal adenocarcinoma in obesity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, THE 16-kDa peptide hormone product of the ob gene, is secreted predominantly by adipose tissues and circulating levels are directly proportional to body fat mass, and so levels are increased in obesity (1). The effects of leptin on body weight and peripheral energy expenditure are well described (2), but it has become apparent that leptin has a plethora of other activities including regulating angiogenesis, wound healing, fertility, immune function, and renal and lung functions (3, 4, 5, 6).

Leptin exerts its effects through interaction with the specific cell membrane localized receptor. There appear to be at least six different receptor variants (Ob-Ra through Ob-Rf), all generated by differential splicing of the db leptin receptor gene product. All receptors share the same extracellular ligand binding domain but differ in the intracellular portion and presumably in the ability to transduce signals. The full-length form (Ob-Rb) is believed to be predominantly responsible for active signal transduction (7), although activation of the MAPK cascade by the Ob-Ra short receptor form has been described (8, 9). The Ob-Rb isoform is extensively expressed in the hypothalamus, in which it mediates the anorectic effects of leptin, but in keeping with the pleiotropic effects of leptin, leptin receptors are widely expressed in peripheral tissues. Typically expression of the Ob-Ra predominates over the other shorter forms in peripheral tissues. The specific actions, if any, of all the isoforms remain unclear.

Recent studies have shown that leptin is a growth factor for a variety of cell types. Leptin stimulated proliferation of hemopoietic cells and breast, colon, gastric, pancreatic ß-, kidney, and lung cell lines in culture (5, 6, 10, 11, 12, 13, 14, 15), and exogenous leptin stimulated colonic proliferation in vivo (10). However, leptin has been reported to reduce the proliferation of the pancreatic PANC-1 and Mia-PaCa cell lines and T24 bladder cancer cells (11, 16).

Esophageal adenocarcinoma (EAC) is increasing in incidence rapidly in the developed world; in contrast, the incidence of squamous cell esophageal cancer remains stable. In 2004 there were more than 14,000 cases of EAC in the United States and more than 13,000 deaths (17). The incidence of EAC has increased by more than 6-fold in the United States over the last 30 yr, and even greater increases have been reported in the United Kingdom (17). The etiology of EAC remains unclear, but the two most important risk factors are obesity and gastroesophageal reflux. The increasing incidence of EAC parallels that of obesity, and detailed case-control studies have confirmed that obesity is a potent risk factor for EAC (18, 19). There is a dose-dependent relationship between body mass index (BMI) and risk of EAC: the odds ratio of developing EAC for those in the upper quartile for body weight is 7.6, compared with those in the lowest quartile and the odds ratio for the obese (BMI > 30 kg/m2) is 16.2, compared with the leanest (BMI < 22 kg/m2) (18). In contrast, there is no relationship between esophageal squamous cancer and body mass. Initial suggestions that the relationship between obesity and EAC was secondary to increased acid reflux in obesity have not been supported by evidence: obesity and acid reflux appear to be independent (18). Whereas there are undoubtedly many possible explanations for this association, it is notable that link between EAC and obesity is stronger than most other cancers, and it is possible that the increased circulating levels of leptin in obesity directly contribute to progression of EAC.

Most cases of OAC develop from metaplastic esophageal columnar epithelium (Barrett’s esophagus). The factors determining progression are not well understood, but compared with normal esophageal epithelium, the metaplastic glandular epithelium has a higher basal proliferative rate and reduced apoptotic index (19). It is believed that these changes are important in the progression to cancer, although the specific factors driving these remain to be determined, the increased proliferation and reduced apoptosis promoting the accumulation and persistence of genetic abnormalities. EAC has not typically been thought of as an endocrine-related cancer, in the manner of breast or prostate cancer, but recent studies have shown that endocrine influences might be important. For example, cholecystokinin2 receptors are expressed by premalignant and malignant esophageal epithelial cells, and gastrin is a growth factor for Barrett’s esophagus and EAC (20). Therefore, we hypothesized that leptin promotes the development of EAC by acting as a growth factor and/or inhibiting apoptosis. We examined the effects of leptin on the OE33 Barrett’s adenocarcinoma cell line. This has been used successfully as an in vitro model for Barrett’s esophagus, results being comparable with human biopsy explants in short-term culture and preliminary data from noncancerous cell lines (21, 22).

We examined the receptor expression, effects on proliferation and apoptosis and early signal transduction events stimulated by leptin in OE33 cells. Previous studies have shown that a wide variety of signaling pathways may be involved in mediating the effects of leptin (7, 23). However, the specific sequence or interactions between these pathways remains to be clarified. Increased activation of the ERK, p38 MAPK, and c-Jun NH2-terminal kinase (JNK) and protein kinase C (PKC) pathways has been described in Barrett’s esophagus, and these may regulate proliferation and apoptosis in EAC and Barrett’s esophagus (19). There is also increased expression of cyclooxygenase (COX)-2 with progression of EAC (24) and overexpression and activation of the epidermal growth factor receptor (EGFR) has been documented (25). Again there is no consensus on the interactions and significance of these signaling pathways in EAC. Therefore, we examined the role of these pathways specifically in the effects of leptin in EAC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant human leptin was purchased from Bachem (St. Helens, UK), SB 203580 (p38 MAPK inhibitor), GF 109203X (PKC inhibitor), SP 600125 (JNK inhibitor), AG 490 [Janus tyrosine kinase (JAK)-2 inhibitor] and GM 6001 [broad-spectrum matrix metalloproteinase (MMP) inhibitor] were purchased from Merck (Nottingham, UK). Indomethacin (nonselective COX inhibitor), the selective COX-2 inhibitors celecoxib and NS-398, LY 294002 [phosphatidylinositol 3'-kinase (PI3-kinase) inhibitor], AG 1478 (EGFR-kinase inhibitor), rapamycin [mammalian target of rapamycin (mTOR) inhibitor], 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP2; src kinase inhibitor), and PD 98059 (p42/44 ERK inhibitor) were from Alexis Biochemicals (Nottingham, UK). 3-[4, 5-Dimethylthiazol-2-y-l]2, 5 diphenyltetrazolium bromide (MTT) and AH 23848 (prostaglandin-EP-4 receptor antagonist) were from Sigma (Poole, UK). Recombinant human IL-1ß was from R&D Systems (Abingdon, UK). All other reagents were from Sigma. When appropriate, stock solutions of inhibitors were dissolved in dimethyl sulfoxide. Inhibitor concentrations were chosen based on published data from our own and others’ laboratories (23, 26). The final concentration of dimethyl sulfoxide in experiments was always less than 0.1%, and control wells contained an equivalent concentration. Inhibitors were added 60 min before the addition of leptin in all experiments unless otherwise stated.

Cell culture and chemicals
The OE33 human Barrett’s-derived esophageal adenocarcinoma cell line was obtained from the European Collection of Cell Cultures (Salisbury, UK) and cultured in DMEM containing 4500 mg/liter glucose, 100 mg/liter penicillin, 100 mg/liter streptomycin, and 2 mM [SCAP]L-glutamine and supplemented with 10% fetal bovine serum (FBS) as previously described (20). All cell culture media and supplements were from Invitrogen (Paisely, UK).

RT-PCR
Total RNA was extracted from cells using a one-step RNA extraction kit (QIAGEN, Crawley, UK) following the manufacturer’s instructions; 1.568 µg RNA was reverse transcribed using previously published custom primers (10, 27). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin primers were gifts from Dr. John Winpenny (University of East Anglia, Norwich, UK); prostaglandin receptor and leptin receptor primers were obtained from Invitrogen. The sequences of all primers and expected product sizes are shown in Table 1Go. Reverse transcriptase comprised incubation at 50 C for 30 min and 95 C for 15 min. PCR was performed for 40 cycles comprising a 45-sec denaturation step at 94 C, a 45-sec annealing step at 58 C, and a 60-sec extension step at 72 C followed by 10 min at 72 C. Electrophoresis on a 0.8% agarose gel stained with ethidium bromide was used to separate PCR products. PCR products were then extracted from the gel using the QIAGEN gel extraction kit, and confirmatory sequencing was performed by the John Innes Centre Genome Laboratory (Norwich, UK).


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  		TABLE 1. Specification of oligonucleotide primer pairs used for RT-PCR assays

 
Western blotting
Cells were grown in 10% FBS-containing medium in a monolayer in petri dishes until confluent. Cells were washed twice in 5 ml of cold PBS and then incubated in 1 ml lysis buffer [containing 150 mM NaCl, 50 mM Tris, 1.5 mM MgCl, 10% glycerol, 1% Nonidet P-40, 1 mM EDTA, 100 µl/10ml phosphatase-inhibitor cocktail (Merck) and protease inhibitors tablet (1/10 ml) (Roche Applied Science, Mannheim, Germany)] on ice in a cold room for 10 min. Protein concentration was quantified using the Bio-Rad Bradford protein assay with bovine serum albumen as standard. Ten micrograms of protein were loaded onto 7.5% SDS-PAGE and blotted onto polyvinylidene difluoride membrane. A COLO 320DM cell lysate (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a positive control for leptin receptor expression. Coomassie brilliant blue staining was used to confirm equal loading of protein samples. Blots were incubated overnight in a cold room in 1:200 antileptin receptor (Ob-R) antibodies (M-18: sc-1834; and H-300: sc-8325, Santa Cruz) or 1:200 anti-EP-4 receptor (H-160: sc-20677, Santa Cruz) in 5% low-fat milk. Immunoreactive proteins were visualized by incubating in 1:2000 horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) in 5% low-fat milk at room temperature on an orbital shaker for 2 h. After washing, membrane was incubated for 5 min in an equal-parts mixture of the SuperSignal West Pico stable peroxide solution and luminol/enhancer solution (Pierce, Northumberland, UK). Chemiluminescence was detected using a Xograph Imaging Systems (Gloucestershire, UK) Compact X 4 machine.

Immunocytochemistry
Cells were seeded onto coverslips in 10% FBS-containing medium. Coverslips were air dried at room temperature before washing in PBS once. Cells were then fixed in 4% formaldehyde for 10 min at room temperature and washed again once in PBS. Fixed cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature followed by three washing steps at room temperature for 5 min each in PBS. Cells were incubated in 2% rabbit serum for 45 min, washed once in PBS for 5 min and then incubated in 1:200 M-18 antileptin receptor antibody in 1% BSA in PBS overnight at 4 C. After further washing in PBS, cells were incubated with 1:400 donkey antigoat fluorescein isothiocyanate (FITC) conjugated secondary antibody (Santa Cruz) for 30 min at room temperature and then washed for 5 min in PBS at room temperature and in low-lighting conditions three times, mounted on slides, and visualized with a Axioplan 2 charge-coupled device upright fluorescent microscope equipped with AxioVision 4.3 digital imaging software (Zeiss, Göttingen, Germany). Images were acquired at x63 magnification.

MTT assay
Cells were seeded at 5 x 104 cells/well in 48-well plates in 10% FBS-containing medium for 24 h and subsequently serum starved for 24 h. Cells were treated with inhibitors and leptin and relative cell numbers after 24 h determined using the MTT colorimetric assay as previously described (28).

Bromodeoxyuridine (BrdU) incorporation assay
A total of 2 x 103 cells/well were cultured in 96-well plates in 10% FBS-containing medium until 60–70% confluent and subsequently serum starved for 24 h. Cells were then treated with 10 nM leptin and 1 µM prostaglandin E2 (PGE2) and incubated for a further 24 h. DNA synthesis and relative cell proliferation were assessed using the BrdU incorporation assay (Roche, Mannheim, Germany). Briefly, cells were incubated with BrdU labeling solution for 2 h at 37 C. After removal of the labeling solution, cells were fixed and denatured and incubated for 90 min with anti-BrdU antibody conjugate, which was subsequently removed by rinsing three times. Finally, cells were incubated in substrate solution at room temperature and proliferation assessed by colorimetric detection.

Assessment of apoptosis
Measurement of intracellular nucleosomes was used for the quantification of apoptosis as described (29, 30). Serum-starved OE33 cells were treated with inhibitors, leptin, or PGE2 for 24 h, and cells were lysed and nucleosomes quantified using the cell death ELISA kit (Roche) according to the manufacturer’s instructions.

COX-2 mRNA assay
A total of 1 x 106 cells/well were seeded into 12-well plates and cultured in complete culture medium for 48 h. After 24 h further culture in serum-free medium, cells were treated with inhibitors and stimulants. Four hours after stimulation, the media were aspirated and cells lysed with cell lysis reagent (R&D Systems). COX-2 and GAPDH mRNA levels in the cell lysates were measured as described previously using Quantikine mRNA ELISA (R&D Systems) according to the manufacturer’s instructions (31).

PGE2 release
Cells were seeded at 1 x 105 cells/well in 24-well plates and cultured in complete medium for 72 h. To test for induction of COX-2 activity, cells were exposed to kinase inhibitors and 10 nM leptin or IL-1ß (10 ng/ml) as a positive control for 18 h before assessment of PGE-2 secretion as previously described using a specific PGE2 ELISA (R&D Systems) (30).

Detection of activated phosphorylated Akt, EGFR, and MAPKs
A total of 104 cells/well were subcultured in 96-well plates for 72 h and subsequently serum starved for 24 h. Cells were then treated with inhibitors for 60 min followed by leptin or PGE2. After exposure to leptin or PGE2, cells were fixed with 4% formaldehyde. Nonphosphorylated total and active phosphorylated Akt, EGFR (tyrosine-1173 phosphorylated), ERK, JNK, and p38 MAPK were quantified using the FACE ELISA kits (Active Motif, Rixensart, Belgium) as described previously (26, 32).

Detection of activated JAK2
Total and tyrosine-phosphorylated JAK2 were quantified in stimulated and formalin-fixed cells using a specific cell-based ELISA as previously described (26).

Statistical analysis
Proliferation studies were done in triplicate or quadruplicate wells. COX-2 mRNA and bioactivity studies were performed in duplicate wells with each well assayed in duplicate. Experiments for direct detection of other signaling intermediates were performed in triplicate wells. The mean of all duplicates from one experiment were regarded as n = 1. Each experiment was repeated three to eight times. Results are expressed as mean ± SEM, and effects were compared with untreated control cells on the same plate. One-way ANOVA was used for dose-response curve and paired t tests were used to analyze the effect of inhibitors. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leptin receptors are expressed by OE33 cells
Expression of the long, short, and common variants of the leptin receptor mRNA in OE33 cells was confirmed by RT-PCR using specific primer pairs (Fig. 1AGo). Correct identification of the sequences was confirmed by sequencing. The mRNA band for the long isoform was less prominent than the bands for the short and common variants. Expression of the leptin receptor protein in OE33 cells was confirmed with Western blotting and immunofluorescence microscopy. Three proteins bands of 120, 100, and 60 kDa were consistently detected in OE33 cells, the largest two corresponding to the Ob-Rb and Ob-Ra receptors, respectively (Fig. 1BGo). Ob-Ra expression was relatively greater than Ob-Rb. Similar results were obtained with both the H-300 antibody, which binds to an internal domain (residues 541–840) of the full-length leptin receptor and M-18 antibody, which is directed against the C-terminal region of the Ob-Ra isoform sequence. Strong cell surface immunoreactivity was detected using FITC immunofluorescence microscopy (Fig. 1CGo). Immunofluorescence was absent in control cells that had not been treated with anti-Ob-R antibodies (data not shown).


Figure 1
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  		FIG. 1. OE33 cells express long and short forms of the leptin receptor and leptin stimulated proliferation and inhibited apoptosis in OE33 cells. A, Expression of mRNA for the long (Ob-Rb), short (Ob-Ra), and common leptin receptor forms was detected by RT-PCR using specific primers. Representative results from one of three experiments are shown. B, Immunoblotting with anti-Ob-R antibody (H-300) with OE33 cells, compared with COLO320DM as positive control. Results are representative of three experiments. C, FITC immunofluorescence for Ob-R expression. OE33 cells were treated with M-18 anti-Ob-R antibody and immunofluorescence performed after binding FITC-donkey antigoat secondary antibody. Negative control, without primary antibody, showed no specific receptor expression (not shown). Results are representative of three different experiments, with at least six slides examined for each experiment. D, Serum-starved OE33 cells were stimulated for 24 h with increasing concentrations of leptin. Relative cells number was assessed by MTT assay, n = 6. E, Serum-starved OE33 cells were stimulated for 24 h with 10 nM leptin. DNA synthesis was assessed by BrdU colorimetric assay, n = 3. F, Serum-starved OE33 cells were treated with 10 nM leptin for 24 h. Apoptosis was quantified by ELISA of intracellular nucleosomes, n = 8. Results expressed as mean ± SEM. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

 
Leptin stimulates OE33 cell proliferation and inhibits apoptosis
Leptin produced a dose-dependent increase in cell number over the concentration range 0.1 pM to 10 nM (Fig. 1DGo). Reliable maximal effects were produced by 10 nM, and this concentration was used for further characterization. To substantiate that the increase in cell number detected with the MTT assay was due to increased proliferation, we confirmed that leptin stimulated DNA synthesis as measured by a BrdU incorporation assay. In this case 10 nM leptin stimulated DNA synthesis by 43 ± 5% (Fig. 1EGo). Having determined the concordance between the assays, the MTT assay was used in preference for further studies because of cost and convenience. Leptin also significantly inhibited serum starvation-induced apoptosis by 39 ± 9% (Fig. 1FGo).

Leptin-induced proliferation of OE33 cells is ERK, p38 MAPK, JNK, JAK2, PI3-kinase/Akt, COX-2, and EGFR dependent but mTOR independent
Leptin-induced proliferation was abolished by the selective COX-2 inhibitors NS-398 and celecoxib as well as the nonselective COX inhibitor indomethacin (Fig. 2AGo). Pharmacologic inhibition of ERK with PD 98059, JAK2 with AG490, and the PI3-kinase/Akt pathway with LY 294002 also abolished leptin-induced proliferation. Inhibition of p38 MAPK with SB 203580 and JNK with SP600125 inhibited leptin-induced proliferation. Pretreatment of cells with the EGFR kinase inhibitor AG1478 also significantly inhibited leptin-induced proliferation, but leptin-induced proliferation was unaffected by pretreatment of cells with rapamycin, the specific mTOR inhibitor (Fig. 2BGo).


Figure 2
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  		FIG. 2. Intracellular pathways involved in the growth promoting and antiapoptotic effects of leptin. OE33 cells were treated with COX inhibitors; indomethacin (Indo; 10 µM); NS398 (5 µM) or celecoxib (1 µM; A and C); or kinase inhibitors, PD98059 (PD; 25 µM), SB 203580 (SB; 5 µM), SP 600125 (SP; 10 µM), LY294002 (LY; 10 µM), AG490 (25 µM), AG1478 (250 nM), or rapamycin (Rapa; 100 nM) before treatment with leptin (10 nM for 24 h) (B and C). Relative cell numbers (A and B) were assessed by MTT assay and apoptosis by intracellular nucleosome ELISA. Results expressed as mean ± SEM, n = 3–6. *, P < 0.05 vs. leptin; **, P < 0.01 vs. leptin; ***, P < 0.01 vs. untreated control.

 
Leptin inhibits apoptosis in OE33 cells via COX-2, ERK, p38 MAPK, JNK, Akt, and EGFR-dependent pathways
The inhibition of apoptosis by leptin was effectively reversed by the selective COX-2 inhibitor NS-398 as well as the specific inhibitors of ERK, p38 MAPK, JNK, PI3-kinase/Akt, and the EGFR (Fig. 2CGo).

Leptin-induced COX-2 bioactivity in OE33 cells is dependent on activation of ERK, p38 MAPK, JAK2, and Akt but not mTOR, JNK, and EGFR kinase
Leptin increased secretion of PGE2 by OE33 cells by 163% (Fig. 3AGo). In this regard leptin was almost as effective as IL-1ß used as positive control (191% increase). The effect of leptin was blocked by the selective COX-2 inhibitor NS-398 and also by inhibition of ERK, p38 MAPK, PI3-kinase, and JAK2 (Fig. 3AGo). However, inhibition of JNK with SP600125 or EGFR with AG1478 did not reduce leptin-stimulated PGE2 production. Similarly rapamycin did not alter leptin-stimulated PGE2 release (Fig. 3AGo). To confirm the results of the PGE2 release experiments, we then assessed the effect of 4 h treatment with leptin on COX-2 mRNA expression. Leptin increased COX-2 mRNA levels by 112%. Pretreatment of cells with inhibitors of ERK, p38 MAPK, JAK2, and PI3-kinase/Akt abolished induction of COX-2 mRNA by leptin, but the inhibitors of JNK and EGFR failed to affect leptin-stimulated COX-2 mRNA levels (Fig. 3BGo).


Figure 3
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  		FIG. 3. Leptin stimulated PGE2 production and COX-2 mRNA expression in OE33 cells. Serum-starved OE33 cells were treated with 10 nM leptin for 18 h (for PGE2 production, A, top) or 4 h (for mRNA quantification, B, bottom). Inhibitors were added 60 min before leptin (PD98059, 25 µM; SB 203580, 5 µM; SP 600125, 10 µM; LY294002, 10 µM; AG490, 25 µM; AG1478, 250 nM; or rapamycin, 100 nM), except for NS-398 (5 µM), which was added 60 min before PGE2 assay. A, PGE2 was quantified by a specific ELISA, results expressed as picograms secreted per 30 min, mean ± SEM, n = 3. B, COX-2 and GAPDH mRNA levels were quantified by specific ELISAs. Results expressed as COX-2 expression relative to GAPDH, compared with untreated control cells, mean ± SEM, n = 3. *, P < 0.01 vs. basal; **, P < 0.05 vs. leptin-treated; ***, P < 0.01 vs. leptin-treated.

 
Leptin stimulated immediate phosphorylation of ERK, p38 MAPK, JAK2, and Akt but not JNK or EGFR
Stimulation of OE33 cells with leptin led to an immediate increase in ERK phosphorylation. Five minutes after exposure to leptin, ERK phosphorylation had increased by 147%. Similarly, leptin increased significantly immediate phosphorylation of p38 MAPK (by 44%), Akt (by 166%), and JAK2 (by 178%). However, leptin failed to increase immediate phosphorylation of JNK or the EGFR (Fig. 4Go). Time-course studies show no significant increase in either JNK or EGFR phosphorylation between 2 and 30 min after leptin stimulation. The specific inhibitors of each kinase blocked leptin-stimulated phosphorylation of that kinase. The JAK2 inhibitor AG490 blocked phosphorylation of JAK2 and also abolished leptin-stimulated activation of ERK and Akt. However, AG490 did not affect leptin-stimulated p38 MAPK activation (Fig. 4Go). The specific inhibitors of ERK, Akt, p38 MAPK, JNK, and EGFR did not reduce the immediate phosphorylation of any other kinase studied (n = 3, data not shown). NS-398, the specific COX-2 inhibitor, did not affect the immediate activation of ERK, p38 MAPK, Akt, or JAK2 (n = 3, data not shown).


Figure 4
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  		FIG. 4. Activation of signaling kinases by leptin in OE33 cells. Serum-starved OE33 cells were treated with 10 nM leptin and incubated at 37 C for 3 min. Cells were then formalin fixed and ERK (A), p38 MAPK (B), JNK (C), JAK2 (D), Akt (E), and EGFR (F) activation quantified by cell-based specific ELISAs. Inhibitors were added 60 min before leptin (PD98059, 25 µM; SB 203580, 5 µM; SP 600125, 10 µM; LY294002, 10 µM; AG490, 25 µM; AG1478, 250 nM). Results are expressed as ratio of phosphorylated kinase to total kinase, compared with untreated control cells, mean ± SEM, n = 3. *, P < 0.01 vs. basal; **, P < 0.01 vs. leptin-treated.

 
OE33 cells express functionally active EP-4 receptors
RT-PCR demonstrated mRNA expression of only the prostaglandin EP-4 receptor. No expression of EP receptors 1–3 were seen using specific primers under the same conditions (Fig. 5AGo). Expression of EP-4 receptor protein was confirmed by immunoblotting (Fig 5BGo). We examined the functional involvement of the EP-4 receptor using the specific pharmacological antagonist AH23848 (27). PGE2 (1 µM) itself significantly stimulated proliferation of OE33 cells by 104% above control assessed by the MTT assay and 73% when measured using BrdU incorporation (Fig. 5Go, C and D). PGE2 also significantly reduced OE33 cell apoptosis, reducing serum starvation-induced apoptosis by 46% (Fig. 5EGo). The stimulation of proliferation and inhibition of apoptosis induced by PGE2 were both blocked by AH23848 (Fig. 5Go, D and E). The EP-4 antagonist also effectively blocked the growth-promoting effect of leptin (Fig. 5DGo).


Figure 5
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  		FIG. 5. OE33 cells express functionally active PGE2-EP4 receptors. A, RT-PCR was performed on total RNA extracted from OE33 cells using specific primers for all four EP receptors (EP-1 to EP-4). Gel shown is representative of three separate experiments. B, Immunoblotting of OE33 cell lysates with anti-EP-4 antibody (H-160). C, Serum-starved OE33 cells were treated with PGE2 (1 µM) for 24 h and cell proliferation quantified using a BrdU incorporation of ELISA, results expressed as mean ± SEM, compared with untreated control, n = 3. D and E, Serum-starved cells were treated with the EP-4 antagonist AH23848 (10 µM) for 60 min and then stimulated with leptin (10 nM) or PGE2 (1 µM) for 24 h. Relative cell number was assessed by MTT assay (D) and apoptosis by intracellular nucleosome ELISA (E). Results are expressed as mean ± SEM, compared with untreated control, n = 3–6. **, P < 0.01 vs. untreated control; ***, P < 0.01 vs. stimulated cells in absence of AH23848.

 
Leptin induces late activation of EGFR kinase and JNK in a COX-2-dependent manner
Having determined that the growth stimulatory and antiapoptotic actions of leptin were dependent on EGFR and JNK activation, that leptin did not stimulate immediate phosphorylation of either kinase, and that COX-2-derived prostaglandins were downstream mediators of the effects of leptin, we investigated whether prostaglandin-mediated activation of these pathways occurred as a late effect. Significant increases in phosphorylation of both EGFR (Fig. 6AGo) and JNK (Fig. 6BGo) were seen 6 h after initial stimulation with leptin. Inhibition of COX-2 with NS-398 and antagonism of the EP-4 receptor blocked this late kinase activation. Inhibition of EGFR kinase activity with AG1478 abolished the late leptin-induced phosphorylation of both EGFR and JNK, but the specific JNK inhibitor SP600125 did not affect EGFR phosphorylation, suggesting that JNK was activated downstream of the EGFR. Consistent with these data showing that activation of EGFR and JNK are delayed events in the signal transduction pathway, adding the EGFR kinase inhibitor 60 min after the leptin was as efficacious in blocking leptin-induced proliferation as pretreating the cells before adding leptin (Fig. 6CGo).


Figure 6
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  		FIG. 6. Delayed prostaglandin-mediated activation of EGFR and JNK contributes to the proliferative effects of leptin. Serum-starved OE33 cells were pretreated with inhibitors (NS-398, 5 µM; AH23848, 10 µM; AG1478, 250 nM; and SP600125, 10 µM) for 60 min and then stimulated with 10 nM leptin. After 6 h cells were formalin fixed and EGFR (A) and JNK (B) phosphorylation quantified by cell-based-specific ELISAs. Results are expressed as phosphorylated kinase to total kinase, compared with untreated control cells, mean ± SEM, n = 4. *, P < 0.01 vs. untreated control; **, P < 0.01 vs. leptin in absence of specific inhibitor. C, Serum-starved OE33 cells were stimulated with 10 nM leptin and proliferation at 24 h assessed by MTT assay. The EGFR kinase inhibitor AG1478 (250 nM) was added either 60 min before leptin or 60 min after the addition of leptin. Results are expressed as relative cell number, compared with untreated control, mean ± SEM, n = 3. *, P < 0.01 vs. untreated control; **, P < 0.01 vs. leptin in absence of specific inhibitor.

 
PGE2 activates JNK via src-, PKC-, and MMP-dependent transactivation of EGFR
To confirm the involvement of JNK and EGFR pathways distal to PGE2 production, we examined the effect of PGE2 stimulation on phosphorylation of these kinases. PGE2 increased EGFR phosphorylation by 148% (Fig. 7AGo) and JNK phosphorylation by 194% (Fig. 7BGo). The COX-2 inhibitor NS-398 did not affect these effects of PGE2, consistent with COX-2 being upstream of PGE2 in the sequence. The JNK inhibitor SP600125 abolished PGE2-stimulated JNK activation but did not affect EGFR phosphorylation, and the EGFR inhibitor AG1478 blocked activation of both EGFR and JNK, again consistent with JNK being activated downstream of the EGFR (Fig. 7Go, A and B).


Figure 7
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  		FIG. 7. PGE2 stimulates JNK activity via transactivation of the EGFR. Serum starved OE33 cells were treated with inhibitors for 60 min (NS-398, 5 µM; AG1478, 250 nM; SP600125, 10 µM; PP2, 10 µM; GM6001, 25 µM; GF109203X, 1 µM) before stimulation with either PGE2 (1 µM) or leptin (10 nM). After 5 min stimulation, cells were formalin fixed and phosphorylation of EGF (A) and JNK (B) quantified by specific cell-based ELISAs. Relative cell numbers were quantified by MTT assay at 24 h (C). Results are expressed as mean ± SEM, n = 3. *, P < 0.05 vs. leptin- or PGE2-treated cells in the absence of specific inhibitor. Kinase data are expressed as phosphorylated to total kinase, compared with untreated control, cell proliferation as percentage of untreated control cells.

 
The mechanisms of PGE2-stimulated transactivation of the EGF receptor were further dissected with specific pharmacological inhibitors. The specific inhibitor of src kinase activity PP2 significantly inhibited phosphorylation of EGFR (Fig. 7AGo) and JNK (Fig. 7BGo), and similar effects were seen with the specific PKC inhibitor GF109203X and the nonspecific MMP inhibitor GM6001. Consistent with these data, PP2, GF109203X, and GM6001 all significantly inhibited both leptin and PGE2-stimulated OE33 cell proliferation (Fig. 7CGo). Inhibition of COX-2 with NS-398 inhibited leptin but not PGE2-stimulated proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we present the novel finding of leptin-stimulating proliferation and inhibiting apoptosis via receptor-mediated signaling in the Barrett’s derived OE33 cell line. This has important implications both for understanding signal transduction of leptin signals in the gastrointestinal tract and determining signals driving malignant progression in Barrett’s esophagus. EAC is becoming an increasingly common disease, and understanding the cellular pathways provides a means to target chemopreventive and treatment options. Our results provide a potential direct link between obesity and OAC: adipose tissue secreting leptin, which promotes growth and inhibits apoptosis.

OE33 cells express mRNA and protein for both the full-length Ob-Rb and shorter Ob-Ra isoforms. These appear to be functional as demonstrated by the biological activity of leptin in regulating proliferation, apoptosis, and kinase phosphorylation. To our knowledge this is the first demonstration of functional leptin receptors in OAC or Barrett’s esophagus. This gives support to our original hypothesis linking obesity and OAC and supporting our conclusions about signaling distal to the Ob-R. A recent preliminary study has reported increased expression of Ob-Rb detected by real-time PCR and immunohistochemistry in Barrett’s esophagus and EAC: expression increasing with malignant transformation from normal to cancer tissues (33). Whereas further studies examining the expression of leptin receptors in EAC are warranted, these data do support the idea that leptin may be a pathogenic factor in EAC. Similarly leptin has been reported to stimulate the growth of two other EAC cell lines (SEG-1 and BIC-1), although that study did not examine any dose-response relationships, receptor expression, or signaling events (34). Leptin was not found to have antiapoptotic actions in that study, but proliferation and apoptosis experiments were performed in serum-containing media, which may have blunted the proliferative and antiapoptotic effects.

The dose-response curve for leptin reported in this study corresponds to the binding affinity of the Ob-R and proliferative responses reported in other model systems (32, 35), consistent with a receptor-mediated effect. The leptin concentrations stimulating cell growth correspond to the physiological circulating concentrations seen in obesity (1). Leptin is also secreted by chief cells into gastric juice (36); it is possible, although speculative at present, that leptin in esophagogastric refluxate also contributes to growth promotion and apoptosis inhibition in vivo.

From our data it is not possible to determine whether the Ob-Rb or Ob-Ra is responsible for signaling in OE33 cells. Both receptors are expressed, although mRNA and protein expression of the shorter form was more prominent. The Ob-Rb has a full-length cytoplasmic domain containing all the motifs and tyrosine residues required for activation of the JAK/signal transducer and activator of transcription (STAT) pathways and has generally been regarded as the fully functional receptor (7). However, signaling via JAK2, ERK, PI3-kinase, and insulin receptor substrate-1 has been documented by Ob-Ra (8). Although activation of STAT proteins is important in mediating many of the biological effects of leptin and activation of STAT3 in the stomach by leptin has been reported (14), we have not specifically examined activation of STAT or other transcription factors because there are no data currently available concerning the involvement of these pathways in EAC, although in other models and disease states, STAT3 appears to be oncogenic and increases the transcription of genes such as survivin, Bcl-xl, and c-fos that may be important in carcinogenesis (37). In contrast, in vivo and in vitro studies have all suggested that the MAPKs, Akt, and COX-2 pathways are involved and important in the pathogenesis of EAC, although the interrelationship of these pathways has not been determined (19, 22, 29, 38). Although we have illustrated the sequence of early signaling events induced by leptin, it must be remembered that other as-yet-uncharacterized pathways may be involved. Because JAK2 is the predominant upstream activator of STAT3, it is possible that STAT3 is activated by leptin in EAC, and it is clear that there is considerable potential for cross-talk between leptin-activated pathways. Serine phosphorylation of STAT3 by MAPKs alters transcriptional activity and STAT3 cooperates with several transcription factors activated by the MAPK cascades (39, 40). Therefore, further studies examining the role of leptin-stimulated STAT protein activation in EAC are likely to prove fruitful.

Previous studies have shown than the proproliferative effects of leptin in HT-29 colon cancer cells and MKN28 gastric cancer cells were ERK dependent (10, 14). Similarly in the MDCK kidney cell line, leptin-induced invasion was dependent on ERK, PI3-kinase, JAK2, and mTOR, although the sequence of these effects was not determined (23). The antiapoptotic effect of leptin in HT-29 cells was ERK dependent, and pharmacological inhibitor studies in SK-N-SH-SY5Y neuroblastoma cells showed that the antiapoptotic effect of leptin involved JAK2, ERK, and PI3-kinase (32, 41).

In this study we have shown that a sequence of signaling events is initiated by leptin and so promotes growth and inhibits apoptosis. Leptin rapidly stimulated phosphorylation of JAK2, ERK, p38 MAPK, and Akt, and all of these kinases were essential to the effects of leptin. In contrast, although JNK and EGFR were essential to the actions of leptin, activation of these appeared to be a later event in signaling: no immediate phosphorylation of these kinases was seen after leptin exposure, phosphorylation was detectable 6 h after stimulation, and the inhibitor of EGFR was effective at abolishing proliferation, even if added 1 h after the leptin. We demonstrated that leptin increased COX-2 mRNA and increased PGE2 production. Inhibitor studies showed that activation of COX-2 and prostaglandin production was an essential event in signal transduction: JAK2, Akt, p38 MAPK, and ERK were upstream of COX-2 and EGFR and JNK were downstream.

Our studies also confirmed that JAK2 was required for activation of the ERK and PI3-kinase/Akt pathways but that activation of p38 MAPK appeared to be JAK2 independent. In gastric cancer cells, JAK2, tyrosine phosphorylation, and the scaffold function but not the phosphatase action of SHP-2 were required for leptin-induced ERK activation (14), and such a pathway could be responsible for ERK and PI3-kinase activation in OE33 cells. The link between Ob-R and p38 MAPK activation remains to be determined. Signaling via JAK1 by the leptin receptors has been demonstrated, and further studies confirming the kinases, small GTPase proteins, and scaffold proteins linking signal transduction pathways may determine further potential therapeutic targets.

Previous data from in vivo and in vitro studies have suggested that ERK, p38 kinase, and Akt are important proliferative and antiapoptotic signals in EAC (29, 38). The precise downstream targets remain to be elucidated: our study suggests that COX-2 is an essential downstream target for all three kinases. Although activation of mTOR and subsequently regulation of transcription via ribosomal p70 S6 kinase has been reported as an important action of Akt, the lack of effect of the specific mTOR inhibitor rapamycin suggests that this pathway is not an essential downstream target of leptin in OE33 cells. Although when activated all three MAPKs and Akt translocate to the nucleus and can phosphorylate a variety of transcription factors, our data do not allow us to determine whether the predominant activities are in increasing COX-2 transcription or increasing mRNA stability. A study has shown that the proliferative responses of acid in esophageal SEG-1 cells are mediated via ERK and p38 MAPK, leading to increased COX-2 transcription (42). However, it is quite clear that COX-2 mRNA levels and bioactivity are increased after leptin treatment. Experimental and observation data have implicated COX-2 in the progression of EAC (19). COX-2 is up-regulated in the transition from normal to premalignant to malignant EAC, Barrett’s esophageal epithelium secretes more PGE2 than normal esophageal epithelium, PGE2 stimulates proliferation of Barrett’s biopsy explants, and COX-2 inhibitors reduce EAC cell proliferation and increase apoptosis in vitro and in vivo (22, 24, 43, 44).

There are relatively few data concerning leptin and COX-2 activation. In rabbit oviduct leptin has been reported to increase PGF2{alpha} but decrease PGE2 (45), and in macrophage J774A.1 cells, leptin increased COX-2 expression and PGE2 production (46). Our data show that COX-2-derived prostaglandins are essential to the proliferative and antiapoptotic effects of leptin. Although COX-2 is a downstream mediator of the growth factor effects of several hormones, to our knowledge this is the first demonstration of the involvement of this pathway in the effects of leptin. Although COX-2 is up-regulated in Barrett’s esophagus and EAC, this does not appear to be due to the inflammatory response (47), our data suggest that leptin may be an important factor in driving COX-2 expression in vivo.

Prostaglandins have a variety of procarcinogenic actions, but there a very limited data concerning the cellular actions of prostaglandins in EAC. Our data suggest that in OE33 cells the downstream effects of PGE2 are mediated via the EP-4 receptor and involve activation of EGFR and JNK. The effects of leptin were replicated by adding exogenous PGE2, and the effects of both leptin and PGE2 were abolished by the specific EP-4 receptor antagonist. Using RT-PCR, we were able to detect expression of the EP-4 receptor type only in OE33 cells, and immunoblotting confirmed expression on EP-4 receptor protein, consistent with this receptor mediating the effects of leptin. There are very limited data on EP-receptor expression in OAC; one study in a rat model showed increased expression of EP-2, EP-3, and EP-4 by RT-PCR. Although not truly quantitative, EP-4 seemed to be increased the most (48), but clearly further studies in humans are required.

In recent years it has been reported that transactivation of EGFR can occur secondary to activation of several hepatohelical G protein-linked receptors. Such an effect is important in mediating the growth-promoting effects of gastrin via the cholecystokinin2 receptors, acetylcholine at muscarinic receptors, and prostaglandins (28, 49, 50, 51). A variety of methods seems to be involved in this effect including cleavage of membrane bound EGFR ligands leading to autocrine and paracrine activation of the EGFR as well as more direct enzymatic tyrosine phosphorylation of the intracellular portion of EGFR (49). In this study we have shown that the broad spectrum MMP inhibitor GM6001, the PKC inhibitor GF109203X, and the src inhibitor PP2 all inhibited the proliferative effects of leptin and PGE2 as well as blocked EGFR phosphorylation. This suggests that all these transduce signals between the EP-4 receptor and EGFR, although further studies are required to dissect this further. Cleavage of EGFR ligands stimulated by PKC and mediated by MMPs has been reported, and we believe that this is the likely mechanism in this case, although further studies concerning the specific ligands are needed (28). Although leptin has been shown to increase the production of various MMPs, this is the first study to implicate MMP activity in the growth-promoting and antiapoptotic effects of leptin (52). The specific MMPs responsible require determination. The predominant MMP expressed in EAC is MMP-7, but clearly this mechanism may provide further therapeutic targets (53). Recently leptin was reported to stimulate immediate tyrosine phosphorylation of the EGFR in gastric cancer cell lines; this mechanism appears to be separate from the one we have described and suggests that there are alternate possible tissue or tumor-specific routes of EGFR activation (54).

Both the EGFR and receptor ligands are increased with malignant progression in Barrett’s esophagus and EAC (25, 55), once again suggesting how leptin can be involved in driving progression in EAC. Our data show that activation of JNK is a necessary downstream target of EGFR activation. Leptin stimulated late phosphorylation of JNK in a COX-2-, EP-4-, and EGFR-dependent manner, and PGE2 stimulated early JNK phosphorylation, again dependent on EP-4 and EGFR kinase activity. In contrast, the specific JNK inhibitor did not affect EGFR phosphorylation. Activation of JNK has been reported to have both pro- and antiapoptotic effects in different models, probably dependent on which specific downstream targets are phosphorylated (26). The predominant target of JNK is phosphorylation and activation of c-Jun as part of activator protein-1 dimers, and this is probably important in the proliferative and antiapoptotic effects; however, JNK has also been reported to regulate apoptosis by regulating the function of Bax and Bcl-2 (56, 57). In SEG-1 OAC cells, acid exposure and subsequent neutralization caused JNK activation that was important in the proliferative response, although there was no indication of any involvement of prostaglandins and the EGFR in this immediate response (29). Although we have described a sequence of events secondary to leptin activating the Ob-R, leading eventually to JNK activation, our present data do not allow us to conclude than JNK is the final common pathway, although clearly this whole complex cascade is essential to the biological activities. It is that possible that continued activation of the other MAPKs or Akt secondary to either direct Ob-R or subsequent EGFR activation is also required as part of the ongoing portfolio of signaling promoting growth and inhibiting apoptosis.

The main aim of our study was to examine the early signaling events stimulated by leptin regulating proliferation and apoptosis. We have not yet examined the main downstream effectors of these leptin (and PGE2) mediated effects. Further studies will be required to determine the alterations in gene expression and protein activity regulating the cell cycle and apoptosis. Our study could be criticized for our reliance use of pharmacological inhibitors and detection of kinase phosphorylation as a marker of enzyme activity. However, we have used widely used, potent, and specific inhibitors of the respective kinases, and detection of specific phosphorylated kinases (whether by ELISA or immunoblotting) is widely used as reliable surrogate read-out of kinase activity. We have demonstrated that leptin induced biological effects (inhibition of apoptosis, stimulation of proliferation, increased COX-2 mRNA), that these were associated with increases in the active forms of the implicated kinases, and that the pharmacological inhibitors specifically abolished phosphorylation of the kinases and blocked the biological end responses. Thus, we feel our data are robust enough to support our conclusions about the involvement of the signaling pathways.

In view of the poor prognosis of EAC, new therapeutic agents are required both for prevention and as sensitizers for current therapies. The complexity of the pathways induced by leptin also suggests several new points at which chemopreventive or therapeutic agents may be targeted. The involvement of COX-2 suggests that inhibition of the pathway might be beneficial in obese patients. Natural plant products that inhibit Akt such as derivatives of quercetin and deguelin may also be potentially useful as part of a dietary approach (58, 59).

In conclusion, we have demonstrated receptor-mediated stimulation of growth and inhibition of apoptosis by leptin in esophageal adenocarcinoma cells. These effects were mediated via a complex cascade of reactions. Initial stimulation with leptin stimulates JAK2 activation and subsequent activation of ERK and Akt; leptin also stimulated rapid activation of p38 MAPK. Increased COX-2 mRNA and PGE2 production occur secondary to ERK, Akt, and p38 MAPK activation. Subsequent activation of the prostaglandin EP-4 receptor leads to transactivation of the EGFR via MMP-, src-, and PKC-dependent pathways and activation of JNK (Fig. 8Go). These pathways are essential to the effects of leptin and may provide a biological link between obesity and the increased proliferation and reduced apoptosis seen in Barrett’s esophagus and EAC.


Figure 8
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  		FIG. 8. Schematic representation of pathways involved in leptin-stimulated cell proliferation and inhibition of apoptosis in OE33 EAC cells. Stimulation of leptin receptors on OE33 cells leads to activation of the janus kinase JAK2, and this in turn mediates activation of ERK and Akt. Subsequent to leptin receptor activation, p38 MAPK is also activated by a JAK2-independent pathway. ERK, Akt, and p38 MAPK are all required to increase COX-2 mRNA levels and consequently increase PGE2 production. PGE-2 activates the EP-4 receptor, which then transactivates the EGFR via a mechanism involving PKC, src tyrosine kinase, and MMPs. JNK is activated downstream of the EGFR and the whole cascade stimulates cell proliferation and inhibits apoptosis. This is a schematic representation and does not imply either that any mediator does not have other roles in cell proliferation or inhibition of apoptosis or that where a sequence of events is shown that this downstream activation is direct.

 


   Acknowledgments
 
We thank Patricia Lunness for technical assistance and Dr. John Winpenny for the ß-actin and GAPDH probes.


   Footnotes
 
This work was supported by The Norfolk and Norwich Hospital Bicentenary Trust Research Student’ship (to O.O.), The Royal Society, The Peel Medical Research Trust, Mason Medical Research Foundation, The Institute of Biomedical Science, and National Health Service Research and Development funding (to I.L.P.B).

Part of this work was presented in abstract form at the United European Gastroenterology Week, Copenhagen, Denmark, 2005, and published in abstract form [Gut 2005;54(Suppl VII):A70].

Disclosure statement: The authors have nothing to disclose.

First Published Online June 1, 2006

Abbreviations: BrdU, Bromodeoxyuridine; COX, cyclooxygenase; BMI, body mass index; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JAK, Janus tyrosine kinase; JNK, c-Jun NH2-terminal kinase; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; MTT, 3-[4, 5-dimethylthiazol-2-y-l]2, 5-diphenyltetrazolium bromide; EAC, esophageal adenocarcinoma; Ob-Ra, short receptor form; Ob-Rb, full-length receptor form; PGE2, prostaglandin E2; PI3-kinase, phosphatidylinositol 3'-kinase; PKC, protein kinase C; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine; STAT, signal transducer and activator of transcription.

Received February 22, 2006.

Accepted for publication May 22, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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