1 Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854,
2 Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030 and
3 Chemoprevention Branch, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20852, USA
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Abstract |
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Abbreviations: AA, arachidonic acid; CLE, columnar-lined esophagus; Cox, cyclooxygenase; DFMO, -difluoromethylornithine; EAC, esophageal adenocarcinoma; EGDA, esophagogastroduodenal anastomosis; EIA, enzyme immunoassay; ESCC, esophageal squamous cell carcinoma; FAP, familial adenomatous polyposis; GERD, gastroesophageal reflux disease; HETE, hydroeicosatetraenoic acid; LC/MS/MS, high performance liquid chromatography/electrospray ionization tandem mass spectrometry; Lox, lipoxygenase; LTB4, leukotriene B4; NDGA, nordihydroguaiaretic acid; NSAIDs, non-steroidal anti-inflammatory drugs; ODC, ornithine decarboxylase; PGE2, prostaglandin E2
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Introduction |
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A large body of evidence has demonstrated a close relationship between aberrant arachidonic acid (AA) metabolism and many types of human cancers. Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs), especially cyclooxygenase (Cox) inhibitors, in rheumatic patients is related to reduced risks of various human cancers, including EAC and esophageal squamous cell carcinoma (ESCC) (5,6). These NSAIDs are believed to exert their chemopreventive effects mainly by inhibiting AA metabolism, although other mechanisms have also been suggested (7,8). Current aspirin users were at a decreased risk for EAC (odds ratio, 0.37; 95% confidence interval, 0.240.58). Risk was similarly reduced among current users of non-aspirin NSAIDs (6). For ESCC that overexpresses Cox2 (9), NSAIDs have shown definite chemopreventive effects in several studies on cell lines and carcinogen-induced animal models (10,11).
The major AA metabolite in normal human esophagus is 12-hydroeicosatetraenoic acid (12-HETE), the metabolite of 12-Lox. Some AA metabolites, such as Cox metabolites (PGE2, D2, F2a), 5-Lox metabolites (LTB4, LTC4) and 15-LOX metabolite (15-HETE), were reportedly increased in GERD patients, especially those with erosive esophagitis (12,13). Acid suppression reduced the production of PGE2 and LTB4 (14) and the cell cycle abnormalities in human CLE (15). Overexpression of Cox2 in human EAC has been confirmed in several studies to date (9,1620). As compared with the normal squamous epithelium of the esophagus, ~4075% CLE, 4080% low-grade dysplasia and 80100% of high-grade dysplasia and EAC overexpress Cox2. This may result from gastroesophageal reflux, especially bile acids, as shown in human EAC cell lines (21) and explant culture of human CLE biopsy tissues (22). Exposure of human esophageal epithelium to both acid and pepsin significantly increased the production of PGE2, suggesting increased Cox2 activity during GERD (23). Clinically, Cox2 overexpression was related to a significant reduction of survival in EAC patients (19). Cox2-specific inhibitors induced apoptosis in human EAC cell lines (24), and reduced the proliferation of CLE cells in human biopsy samples. The addition of exogenous PGE2 reversed the anti-proliferative effect of the Cox2 inhibitor (25). The Lox pathways of AA metabolism are also important in cancer development (26,27). During carcinogenesis, the dynamic balance between the various Lox pathways may shift away from the anticarcinogenic Lox (15-Lox) and toward the pro-carcinogenic Lox(s) (5-, 8- and 12-Lox) (28). All these published data suggest that modulation of AA metabolism, especially inhibition of the Cox, 5-Lox, 12-Lox pathways, may inhibit the formation of EAC.
As a group of molecules essential for cell growth, polyamine levels are often elevated in rodent and human neoplastic cells or tissues (29). Earlier research found the putrescine level increased progressively from gastric fundus, CLE, dysplasia, to EAC, suggesting altered metabolism of polyamines in esophageal adenocarcinogenesis (30). Ornithine deoxycarboxylase (ODC) is known to be elevated in human biopsy CLE samples, compared with adjacent gastric or small intestinal epithelium of the same patients, although putrescine, spermidine, spermine and the spermidine/spermine ratio were not related to the enzyme activity (31). -Difluoromethylornithine (DFMO), a specific ODC inhibitor, either alone or in combination with NSAIDs, has been studied as a cancer chemopreventive agent in preclinical and clinical trials on cancers of different organs including esophagus (29,32,33). In the esophagus, DFMO effectively inhibited N-nitrosomethylbenzylamine-induced ESCC in zinc-deficient rats by stimulating apoptosis and inhibiting cell proliferation (34). DFMO inhibited cell growth of two cell strains derived from human CLE biopsy tissue (31). In a small-scale clinical trial, eight CLE patients were treated with DFMO at a dose of 1.5 g/m2/day for 12 weeks. Spermidine and spermidine/spermine ratio, but not spermine and the ODC activity, in CLE and normal esophageal mucosa were suppressed by DFMO. Polyamine levels recovered to the pre-treatment level when DFMO therapy was discontinued (35).
In this study, we first examined the profile of AA metabolites and Cox2 expression in rat tissues after EGDA. With in situ hybridization, Cox2 expression was examined in esophagitis, CLE and EAC tumors of EGDA rats. Sulindac (a non-specific Cox inhibitor), nordihydroguaiaretic acid (NDGA, a non-specific Lox inhibitor), and DFMO were selected to test their potential efficacy against the formation of EAC in the EGDA rats. Combinations of these agents were also included to test possible synergistic or additive effects on esophageal adenocarcinogenesis.
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Materials and methods |
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The LC/MS/MS method was established for samples of cell lysate and adjusted for tissue samples (36). After homogenization in a buffer containing BHT, NDGA and indomethacin, tissue or cell pellet was extracted with hexane:ethyl acetate under reduced light conditions after the addition of an internal standard (PGE2-d4). The sample was brought to dryness under nitrogen, reconstituted in methanol:2 mM ammonium acetate and analyzed with the established method. To determine the activities of the major AA-metabolizing enzymes or pathways, the tissue homogenate was incubated with exogenous AA before extraction. The extract was analyzed in the same way as described above. The enzyme activities were calculated by subtracting the endogenous metabolites. Since several enzymatic reactions are needed for the biosynthesis of a metabolite, the newly formed metabolite from exogenous AA reflects the activity of certain AA-metabolizing pathways, but not that of a single enzyme. For example, exogenous LTB4 reflected the activities of 5-Lox, 5-Lox activating protein and leukotriene A4 hydrolase, or the activity of this pathway.
In situ hybridization of Cox2
Since the commercially available antibodies used for immunohistochemistry generated staining heterogeneity on our formalin-fixed paraffin-embedded tissue sections, in situ hybridization was used to analyze the expression of Cox2. Digoxigenin-labeled antisense and sense cRNA probes (kindly provided by Dr Xiaochun Xu, M. D. Anderson Cancer Center) were prepared via a digoxigenin-RNA labeling and detection kit (Boehringer Mannheim, Germany) (37). Formalin-fixed paraffin-embedded tissue sections were digested with 10 mg/ml proteinase K, and then hybridized with the digoxigenin-labeled cRNA probe (25 ng/ml) overnight at 37°C in the hybridization buffer. After post-hybridization washing, the hybridization signal was detected as violet by an alkaline phosphatase-linked immunoassay using an anti-digoxigenin antibody. The sense probe was used as the negative control. Paraffin sections of six EGDA rats without iron supplementation were obtained from a previous experiment on EGDA (3).
Short-term dose-selection study
The aim of this short-term experiment was to test the anti-inflammatory effects of sulindac and NDGA in the EGDA model, and help determine proper doses for the long-term chemoprevention study.
Six-week-old male SpragueDawley rats from Taconic Farms (Germantown, NY) were housed two per cage, given AIN93M diet and water ad libitum, and maintained on a 12 h light/dark cycle. They were allowed to acclimate for 2 weeks prior to surgery. Solid food was withdrawn from 1 day before to 1 day after surgery. EGDA was performed according to the procedure described previously (3), which was approved by the Animal Care and Facilities Committee at Rutgers University (protocol no. 94-017). EGDA rats were placed on diets containing the indicated doses of the proposed chemopreventive agents (sulindac 300 and 600 p.p.m. or NDGA 100 p.p.m.) right after receiving EGDA surgery, and remained on the chemopreventive or control diets for a period of 4 weeks (Table II). All the diets were prepared by Research Diets (New Brunswick, NJ). Iron (12.5 mg/kg) was supplemented once during the experiment by i.p. injection at 2 weeks after surgery. Body weight was monitored throughout the experiment. Food and fluid intake were measured at week 1, week 2 and week 4 after surgery. At each time point, three consecutive days were monitored. After the rats were killed, the esophagus was cut longitudinally, fixed in 10% buffered formalin, Swiss-rolled, processed and embedded in paraffin for histopathological analysis.
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Infiltration of macrophages at the squamocolumnar junction was analyzed by immunohistochemical staining for a macrophage/monocyte-specific antigen ED1, and by counting the number of positive cells per high power field (x400). The avidinbiotinperoxidase complex method (Elite ABC kit; Vector Laboratories, Burlingame, CA) and a monoclonal mouse anti-ED1 (Serotec, Oxford, UK; 2 mg/ml) were used for immunohistochemical staining of ED1 according to the product manual. The cells were counted with a computer imaging software ImagePro Plus.
Long-term chemoprevention study
EGDA rats were given the chemopreventive agents in diet or drink after surgery, and iron dextran i.p. (12.5 mg Fe/kg/2 weeks) starting from 4 weeks after surgery (Table III). To assess the effects of the combination of sulindac and NDGA, we used the method of Laska et al. (38) in experimental design. We took 600 p.p.m. sulindac and 200 p.p.m. NDGA as the highest doses. Comparison between the combination group (300 p.p.m. sulindac and 100 p.p.m. NDGA) and the groups with single agents (600 p.p.m. sulindac or 200 p.p.m. NDGA) would tell us whether these two agents are synergistic, additive or antagonistic in their chemopreventive effects. In addition, two more groups with single agents (300 p.p.m. sulindac or 100 p.p.m. NDGA) were included to test for the possible dose-dependent response of sulindac and NDGA.
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Histopathological analysis was carried out on the H&E stained slides: the first, 10th, 20th and 30th slides. CLE was characterized by the presence of intestinal columnar epithelium containing a villiform surface, mucous glands and intestinal-type goblet cells, above the blue prolene suture. Dysplastic lesions were diagnosed by the partial loss of cell polarity and maturation, nuclear atypia and an increase in mitotic figures. EAC was diagnosed when dysplastic columnar epithelial cells invaded through the basement membrane (39).
Enzyme immunoassay (EIA) of PGE2 and LTB4
Eight frozen samples of the rat esophagoduodenal junction from each group of the long-term chemoprevention study were used for this analysis. Frozen tissue samples were analyzed immediately after being taken out of a 80°C freezer. After being homogenized in a buffer containing 0.1 M Tris and 20 mM EDTA (pH 7.1), the tissue samples were aliquoted for determination of protein concentration and organic extraction. The organic extract was dried with nitrogen and reconstituted in the EIA buffer. PGE2 and LTB4 were measured with the experimental procedures provided by the manufacturer of the EIA kits (Cayman Chemical Co., Ann Arbor, MI). The amount of PGE2 and LTB4 were expressed as nanograms per milligram protein.
Statistical analysis
Students t-test was used for most analysis with the computer software Statview 4.2. The results on tumor incidence were analyzed by the 2 test.
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Results |
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Cox2 expression on the esophageal tissues of EGDA rats
With in situ hybridization, we examined the expression of Cox2 on the esophageal tissues of EGDA rats. In the proximal esophagus, Cox2 was not expressed in the epithelial cells. Positive staining was occasionally observed in some stromal cells, presumably inflammatory cells (Figure 1A). In the esophagus with EGDA-induced esophagitis, Cox2 was overexpressed in the basal and parabasal cells of the squamous epithelium. A large of number of infiltrating inflammatory cells also expressed Cox2 (Figure 1BD
). In both CLE and EAC, Cox2 was expressed in the epithelial cells and the infiltrating inflammatory cells (Figure 1B, C, E and F
).
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Short-term experiment
We did not detect any statistical difference in food or fluid intake at weeks 1, 2 and 4 after surgery due to administration of sulindac and NDGA. Fluid consumption decreased slightly in the three experimental groups. No significant difference in body weight was observed among the five groups. No obvious signs of toxicity were observed in the three experimental groups when the histology of the liver and kidney of five rats from each group was examined.
For semi-quantification of inflammation, we focused on the squamocolumnar junction where all the well-differentiated mucinous EAC occurred according to our previous studies. Four aspects of inflammation were examined: inflammatory cell infiltration, basal cell proliferation, capillary in-growth, edema and hyperemia in the interstitial tissue of the esophagus. Both sulindac and NDGA exerted anti-inflammatory effects at the squamocolumnar junction of EGDA rats. Sulindac (600 p.p.m.) was more effective than sulindac (300 p.p.m.), which was not significantly different from NDGA (100 p.p.m.) (Table II).
The number of infiltrating macrophages was used as another parameter of chronic inflammation. With immunohistochemistry and an imaging system, we counted the number of ED1-positive cells at the squamocolumnar junction. Both sulindac and NDGA significantly inhibited infiltration of macrophages/monocytes at the squamocolumnar junction (Table II) (P < 0.05). There was no difference between the two groups treated with sulindac.
Long-term chemoprevention
Most (90%) of the animals survived the surgery and throughout the whole experiment. During the experiment, these animals were active and healthy. In the early time points (up to 24 weeks after surgery), body weights of the experimental groups were slightly lower than the non-operated control (Group I) and the EGDA control (Group II), but the differences were not statistically significant (Figure 2) (P > 0.05). After 24 weeks, the two groups treated with DFMO (Groups VII and VIII) had significantly lower body weight than the non-operated control and EGDA control (P < 0.05). The body weight-lowering effect of DFMO was reported in a previous study using similar doses (40). The body weights of other groups did not decrease significantly as compared with the control groups. In the original experimental design, we had one group treated with 600 p.p.m. sulindac (which was most effective against inflammation in the short-term experiment). Starting at week 4, this group had significantly lower body weight than both control groups (P < 0.05). Gastrointestinal bleeding as detected with an occult blood test kit (Hemoccult slide, SmithKline Diagnostics; San Jose, CA) resulted in the loss of 12 out of 30 rats. This group was thus discontinued at week 11 from this experiment (Figure 2
).
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Sulindac (300 p.p.m.), alone or in combination with NDGA (100 p.p.m.) or DFMO (1%), significantly decreased the tumor incidence (Table III) (P < 0.05). NDGA slightly decreased the tumor incidence, but not significantly (P > 0.05). The effect of 300 p.p.m. sulindac was especially effective when used in combination with 100 p.p.m. NDGA. Since the group treated with 600 p.p.m. sulindac was discontinued, it was not conclusive whether there was any interaction between 300 p.p.m. sulindac and 100 p.p.m. NDGA. DFMO alone did not show significant effects on the tumor incidence (P > 0.05). When used in combination with 300 p.p.m. sulindac, there was significant inhibition of tumor formation (P < 0.05) (Table III
). Although it did not differ significantly from treatment with sulindac alone, presumably the chemopreventive effect was due to sulindac.
As compared with the EGDA control (Group II), 300 p.p.m. sulindac, 200 p.p.m. NDGA, and the two combinations significantly reduced tumor volume in this study (P < 0.05). DFMO alone and 100 p.p.m. NDGA were not effective (P > 0.05) (Table III). This suggests that NDGA inhibits cell growth at high doses. The inhibitory effect has been demonstrated in other tumor models (41).
Effects of sulindac, NDGA and DFMO on the biosynthesis of PGE2 and LTB4 at the esophagoduodenal junction of EGDA rats
To investigate the relationship between AA metabolism and esophageal adenocarcinogenesis, we determined the levels of PGE2 and LTB4 in the esophagoduodenal junction of the EGDA rats (Table III). The esophagoduodenal junction was used because all EAC tumors developed in this region according to our previous studies.
PGE2 increased significantly in the esophagoduodenal junction of all the EGDA rats as compared with the non-operated control (Group I) (P < 0.05). Treatment with 300 p.p.m. sulindac (Group III), 100 p.p.m. NDGA (Group IV) and the combination groups (Groups VI and VIII) had significantly lower PGE2 levels than the EGDA control (Group II) (P < 0.05). The inhibitory effect on PGE2 biosynthesis was stronger when the EGDA rats were treated with the combinations (Table III). The PGE2 level of the non-operated control (Group I) was lower than that in the proximal esophagus of the EGDA rats (Table I
).
Similar to PGE2, LTB4 significantly increased in all the EGDA rats (P < 0.05). However, treatment with sulindac, NDGA and DFMO did not significantly reduce LTB4 biosynthesis in the esophagoduodenal junction, except the combination of sulindac and DFMO (Group VIII) (Table III).
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Discussion |
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Based on the results of LC/MS/MS, some other AA metabolites may also play certain roles in esophageal adenocarcinogenesis, such as LTB4, 5-HETE, 12-HETE, 8-HETE and 15-HETE. Some could be anticarcinogenic, some pro-carcinogenic and some bystanders, as shown in other experimental systems (26,27). Modulation of the related AA-metabolizing enzymes/pathways may be chemopreventive against the development of EAC. This mechanism-based approach, combining biochemical analysis, histopathological analysis and animal studies, is a good approach for chemoprevention studies on esophageal adenocarcinogenesis. Further studies are needed to investigate the roles of other AA-metabolizing enzymes/pathways and their downstream metabolites.
Inflammation at the squamocolumnar junction induced by EGDA was believed to result in aberrant AA metabolism, especially increased PGE2 biosynthesis. As an important event during chronic inflammation, aberrant AA metabolism has long been correlated with carcinogenesis, especially the inflammation-associated cancers. The results from this study support the general hypothesis on the important roles of AA metabolites and oxidative stress in esophageal adenocarcinogenesis (4). It is interesting that the PGE2 level of the non-operated control esophagus was lower than that in the proximal esophagus of the EGDA rats. It is possible that the proximal esophagus was exposed to a certain amount of gastroesophageal refluxate although far away from the anastomosis site. It suggested that PGE2 biosynthesis started to increase before histological inflammation developed. We speculate that inhibitors targeting the Cox/PGE2 pathway could be more effective when used at the earlier stages of cancer development. The results of Cox2 expression with in situ hybridization also supported this hypothesis. Cox2 overexpression was most prominent in the squamous epithelium at the stage of esophagitis. Nevertheless, NSAIDs given later in the esophageal adenocarcinogenesis process may also be effective as suggested in other animal models of skin, colon and bladder cancers (4244).
Sulindac effectively inhibited esophageal adenocarcinogenesis when used alone or in combination with NDGA or DFMO (Table III). During the preparation of this manuscript, Buttar et al. (45) published a chemopreventive study with Cox inhibitors in an animal model similar to EGDA. Both sulindac (30 p.p.m./day) and a Cox2-specific inhibitor (MF-tricyclic, 10 p.p.m./day) reduced the incidence of EAC by 55 and 79%, respectively. Consistent with its inhibition on cancer development, sulindac also reduced the degree of inflammation and Cox activity. A good correlation between the tissue PGE2 level and the risk of EAC was also observed. Both this study and ours suggested that the chemopreventive effect of sulindac was Cox/PGE2-dependent, although Cox-independent mechanisms cannot be excluded. For example, Cox inhibitors (indomethacin, sulindac, celecoxib) inhibited growth and induced apoptosis of human EAC cells in vitro by virtue of their effects on PG biosynthesis (24).
PGE2 can function in both an autocrine manner as well as a paracrine manner to the cells in the vicinity that expresses the specific receptors. Binding of PGE2 to its receptors activates second messengers (cAMP, Ca2+ and inositol phosphates), and induces tumor growth and metastasis (46). These effects have recently been clearly demonstrated in a colon cancer cell line (47). EP1, EP2 and EP4 were all found to play important roles in colon carcinogenesis (4850). Similarly, we found that human EAC cell lines (SEG-1, FLO-1, SKGT4, BIC-1 and BE-3) expressed these EP receptors (data not shown). It is possible that, in our EGDA model, activation of the Cox2 pathway by EGDA-induced reflux increased the biosynthesis of PGE2, which may then bind to the EP receptors to induce hyperproliferation. These cells replaced the squamous epithelium in the esophagus that was destroyed by inflammatory erosion. These combined events facilitate the metaplasia or creeping substitution of squamous epithelium by the columnar epithelium. The mucin-producing nature of the rat EAC may result from hyperproliferation of EP receptor-expressing goblet cells upon stimulation by PGE2 (4).
NDGA, a lignan abundant in the genus Larrea, was used in this study to inhibit 5-, 12- and 15-Lox and P450 monooxygenase (51). NDGA has been shown to inhibit growth and induce apoptosis of various cancer cells (41,52,53), and to inhibit carcinogenesis in animal models of breast and skin cancers (54,55). As a Lox inhibitor, NDGA was especially useful against cancer cell lines that were not sensitive to Cox inhibitors (56). NDGA is also an antioxidant, which might help relieve the oxidative stress in the EGDA model, as suggested by its chemopreventive effect in an oxidative stress-related renal cancer model (57). In this study, NDGA reduced the tumor size at the dose of 200 p.p.m.. Although NDGA alone at the doses of 100 p.p.m. and 200 p.p.m. did not significantly decrease the incidence of EAC, there seemed to be a dose-dependent tendency showing a decrease of EAC incidence from 52.7 (Group II) to 52.4 (Group IV) and 37% (Group V), respectively. When used in combination with 300 p.p.m. sulindac, 100 p.p.m. NDGA reduced the tumor incidence further from 26.9 (Group III) to 16.7% (Group VI), although this was not statistically significant. Since the two doses of NDGA were not toxic to the EGDA rats in this study, it is possible that at higher doses NDGA may be effective against the EGDA-induced EAC.
We did not find a significant effect of NDGA on the 5-Lox pathway as indicated by LTB4 biosynthesis in this study. This may be due to an insufficient dose. Inhibition of 15-Lox may also counteract its inhibitory effect on 5-Lox (58). However, the role of 5-Lox pathway in esophageal adenocarcinogenesis deserves further investigation. In another study, we found overexpression of leukotriene A4 hydrolase, the rate-limiting enzyme downstream to 5-Lox for LTB4 biosynthesis, in rat and human EAC. Bestatin, a specific enzyme inhibitor, significantly reduced the incidence of EGDA-induced rat EAC. Such an effect was correlated with inhibition on the production of LTB4 in rat esophageal tissue (X.Chen and C.S.Yang, manuscript submitted for publication).
DFMO did not significantly inhibit esophageal adenocarcinogenesis. The result was consistent with a recent randomized, placebo-controlled and double-blinded clinical study (59,60). Fifty-two CLE patients were given a placebo or 900 mg DFMO once every day for 6 months. DFMO did not reduce polyamine levels (putrescine, spermidine, spermine and spermidine/spermine ratio) in biopsy CLE tissues. Cell proliferation (Ki-67 labeling index and cyclin D1 labeling index) was not modulated either. It is known that proliferating cells may take up not only endogenous polyamines, but also exogenous polyamines from systemic circulation and gastrointestinal tract (food and bacteria) (61,62). Even during fasting, the gastrointestinal lumen contains a large amount of polyamines, which can be efficiently absorbed by humans and rats (63,64). Moreover, ODC activity can be affected by many factors, including diurnal rhythm, feeding, growth factors and hormones. Since EGDA rats had their duodenum anastomosed to the esophagus, we speculate that a large amount of exogenous polyamines in the upper gastrointestinal tract compromised the effect of DFMO. Based on our findings and others, DFMO may not be a good chemopreventive agent for EAC, especially if DFMO toxicity is considered.
In summary, we demonstrated aberrant AA metabolism in the esophageal tissues of EGDA rats. Increased PGE2 biosynthesis, Cox2 overexpression and chemoprevention with sulindac in the EGDA rats suggested an important role of Cox pathway in esophageal adenocarcinogenesis. For future studies, specific inhibitors of the AA-metabolizing enzymes or metabolite receptors may be tested. Since AA metabolism pathways are closely related with each other in terms of mechanisms of action and substrate preference, blocking one pathway may activate another pathway (65,66). Some cancer cells were sensitive to Cox inhibitors, whereas others were sensitive to Lox inhibitors (67,68). Therefore, combinations of agents targeting different pathways are especially promising in synergistically or additively inhibiting esophageal adenocarcinogenesis and reducing the dose-dependent side effects.
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Notes |
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Acknowledgments |
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References |
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