ARTICLE

Dietary Zinc Modulation of COX-2 Expression and Lingual and Esophageal Carcinogenesis in Rats

Louise Y. Y. Fong, Liang Zhang, Yubao Jiang, John L. Farber

Affiliation of authors: Kimmel Cancer Center and Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA

Correspondence to: Louise Y. Y. Fong, Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107 (e-mail: louise.fong{at}jefferson.edu)


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Cancer of the upper aerodigestive tract, including esophageal and tongue carcinomas, is a major cause of cancer deaths worldwide. Esophageal and tongue cancers have both been associated with dietary zinc deficiency (ZD), and cyclooxygenase (COX-2) is often overexpressed in these cancers. Using rat models, we examined whether zinc regulates COX-2 expression in these cancers. Method: Expression of COX-2 protein and mRNA in rat lingual and esophageal epithelia in control (zinc sufficient [ZS]) rats, during ZD, and after intragastric zinc replenishment (ZR) were determined by immunoblotting, immunohistochemistry, and real-time quantitative polymerase chain reaction. COX-2 gene expression, cell proliferation, and apoptosis were analyzed in ZD, ZR, and ZD rats treated with the COX-2 inhibitors celecoxib and indomethacin. Tumor development in ZD rats treated by continuous exposure to the carcinogen 4-nitroquinoline 1 oxide (NQO), which causes tongue tumors in rats, was compared with those in NQO-treated ZS rats. Statistical tests were two-sided. Results: The esophagus and tongue of ZD rats were hyperplastic and expressed COX-2 protein and mRNA at 8- to 14.7-fold higher levels than control rats. Within hours ZR reduced COX-2 overexpression to threefold that in control rats and reversed the hyperplastic phenotypes. The esophagus of ZD rats treated with celecoxib or indomethacin showed a reduction in cell proliferation and stimulation of apoptosis. NQO treatment resulted in greater incidence of lingual squamous cell carcinomas (74% versus 22%, difference = 52%, 95% confidence interval [CI] = 20% to 80%, P = .015) and greater tumor multiplicity (13.1 versus 4.3, difference = 8.8, 95% CI = 7.0 to 10.6, P = .018) in ZD than ZS rats. Of 23 NQO-treated ZD rats, 39% (9) and 61% (14) harbored esophageal and forestomach tumors, respectively, whereas none of the NQO-treated ZS rats did. Conclusions: COX-2 overexpression accompanies hyperplasia in ZD rats. Increased cell proliferation in NQO-treated ZD rats facilitates the development of tumors at multiple sites. The finding that zinc regulates COX-2 expression in vivo in an animal model may lead to prevention or therapeutic possibilities for upper aerodigestive tract cancer.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Upper aerodigestive tract (UADT) cancer, including esophageal and oral cancer, is an important cause of morbidity and mortality worldwide (1). With a 5-year survival rate of approximately 10%, the prognosis of esophageal cancer is poor. In 2003, 13 900 new cases of esophageal cancer were diagnosed in the United States and 13 000 deaths were attributed to this malignancy (2). The prognosis of oral cancer, the major site being the tongue, is equally dismal. The incidence of oral cancer is increasing worldwide, including in young adults and in people without the documented risk factors of tobacco and alcohol use (3). Patients with oral cancer have a high mortality rate because of field cancerization effects that result in second primary tumors, particularly in the esophagus (46). Thus, chemoprevention strategies that halt or reverse the carcinogenic process are urgently needed.

Zinc deficiency (ZD) is associated with an increased risk for esophageal (79) and oral (10,11) cancer in humans. We have developed ZD rat and mouse esophageal cancer models (1219) and found that ZD creates a precancerous condition in the esophagus and forestomach by causing unrestrained cell proliferation and altering gene expression (14,15). Consequently, ZD accelerates carcinogenesis in the esophagus and forestomach caused by a single exposure to the carcinogen N-nitrosomethylbenzylamine (NMBA) in rats (14,15), in p53-deficient mice (16), and in mice overexpressing cyclin D1 (17). Zinc replenishment (ZR) reverses tumorigenesis by rapidly stimulating apoptosis through Bax expression in the esophageal epithelium (18). In addition, transgenic overexpression of antizyme, a multifunctional regulator of polyamine metabolism that inhibits ornithine decarboxylase activity and polyamine transport, reduces cell proliferation and forestomach carcinogenesis in ZD mice (19). The rapid tumor initiation and reversal in ZD rodent models offers a unique opportunity to both identify relevant biomarkers of UADT cancers in the early stages of carcinogenesis and to test novel chemopreventive and chemotherapeutic strategies for these cancers.

The mechanism by which zinc increases UADT cancer risk is not known, but it is possible that it acts by inducing the activity of cyclooxygenase-2 (COX-2), an enzyme that catalyzes the formation of prostaglandins from arachidonic acid. Whereas COX-1 is constitutively expressed in virtually all mammalian tissues, COX-2 is undetectable in most tissues, but its expression can be induced quickly by factors that have been implicated in carcinogenesis, including growth factors, inflammatory stimuli, oncogenes, and tumor promoters (20). COX-2 is overexpressed in a variety of human premalignant and malignant lesions, including esophageal and oral cancers (2123). Overexpression of COX-2 enhances cell proliferation, inhibits apoptosis (24), modulates cell adhesion and angiogenesis (25), and increases metastatic potential (26), thereby contributing to carcinogenesis. Oshima et al. (27) presented direct genetic evidence that COX-2 plays a key role in tumorigenesis by demonstrating that deficiency of the COX-2 gene in Apc knockout mice greatly reduces intestinal polyp formation. In addition, Apc knockout mice treated with the specific COX-2 inhibitor MF-tricyclic had fewer intestinal polyps than those treated with sulindac, which inhibits both COX-1 and COX-2. These findings provide the rationale for the use of selective inhibitors of COX-2 as a novel class of therapeutic and chemopreventive agents (28).

In this study, we used the ZD rat esophageal cancer model to test the hypothesis that COX-2 expression in esophageal epithelium is modulated by dietary zinc. We extended the ZD rat model to tongue cancer and investigated the responsiveness of the lingual epithelium to zinc. We also investigated the effect of COX-2 inhibitors on the ZD cancer models. Finally, we examined whether ZD rats have an increased risk of developing tongue tumors in response to 4-nitroquinoline 1-oxide (NQO), a carcinogen that causes tongue tumors in rats.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemicals and Animal Diets

NQO was from Wako Chemicals USA (Richmond, VA), celecoxib was from LKT Laboratories Inc. (St. Paul, MN), and indomethacin and polyethylene glycol (PEG) 400 were from Sigma–Aldrich (St. Louis, MO). Custom-formulated, egg white-based ZD and zinc-sufficient (ZS) diets were prepared by Teklad (Madison, WI). The two diets were identical except for the amount of zinc carbonate (3 and 70 ppm for the ZD and ZS diets, respectively).

Experimental Design

The animal studies were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee and were conducted under National Institutes of Health guidelines. Ninety weanling male Sprague–Dawley rats (Taconic, Germantown, NY) were fed a ZD diet ad libitum. Because ZD rats consume less food than ZS rats, 15 additional rats were pair-fed a ZS diet at levels that matched the reduced food consumption of ZD animals (13). All rats had free access to deionized water. After 5 weeks, ZD rats showed evidence of increased cell proliferation (as demonstrated by increased proliferating cell nuclear antigen [PCNA] and 5-bromo-2'-deoxyurdine labeling) in the esophagus (1315). Zinc gluconate (containing 1.2 mg elemental Zn) in 250 µL saline was then administered intragastrically to 75 of the ZD rats, which were immediately switched to the ZS diet, forming the ZR groups. At 2, 8, 12, 24, and 48 hours after replenishment, 15 ZR rats/time point were killed after anesthesia with isoflurane (Datex-Ohmeda Inc., Andover, MA), forming the ZR-2, ZR-8, ZR-12, ZR-24, and ZR-48 groups. Control ZS and ZD rats (15/group) were treated intragastrically with saline lacking zinc and were killed after 8 hours.

Esophageal and tongue epithelia were isolated from all rats for immunoblotting, real-time quantitative polymerase chain reaction (PCR), and immunohistochemistry studies. Esophagi from three to five individual rats from each group were pooled for total RNA preparation. Esophagi from another three to five rats and tongues from two rats from each group were pooled for protein preparation. Esophageal epithelia were prepared by using a blade to strip off the connective tissue/muscle layer (15). Lingual epithelia were prepared as described (29). Briefly, tongue was cut longitudinally into two slices after roughly removing muscle layers and was incubated in a phosphate-buffered saline (PBS)–EDTA buffer containing 100 µg/mL soybean trypsin inhibitor at 37 °C for 2 hours, and then in a fresh buffer at 4 °C overnight. Lingual epithelia were then peeled off from the muscle layers using fine forceps. Lingual and esophageal epithelia were snap frozen in liquid nitrogen and stored at –80 °C until protein or RNA preparation. Immunohistochemistry studies were performed on esophageal and lingual sections from six rats of each group. Esophagi and tongues were fixed in phosphate-buffered formalin, and embedded in paraffin. Serial cross-sections (4 µm) were stained with hematoxylin–eosin or left unstained for immunohistochemical studies.

Protein Extraction and Western Blot

Proteins were extracted from pooled lingual or esophageal epithelia by homogenizing in a buffer containing 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5 mM EDTA, 100µg/mL aprotinin, 50 µg/mL leupeptin, 1 mM benzamidine, 7 µg/mL pepstatin and 1 mM phenylmethylsulfonyl fluoride. Debris was removed by centrifugation at 16 000g for 20 minutes. The protein concentration in the lysates was measured using a Bradford protein assay kit (Bio-Rad, Hercules, CA). Proteins (100 µg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose membranes (15). Membranes were probed sequentially with rabbit polyclonal antisera against COX-2 (Cayman Chemical, Ann Arbor, MI; 1 : 3000 dilution in Super Block blocking buffer [Pierce Chemical, Rockford, IL] containing 0.5% Tween-20), COX-1 (Santa Cruz Biotechnology, Santa Cruz, CA; 1 : 2000 dilution), and GAPDH as a loading and transfer control (Abcam, Cambridge, UK, 1 : 80 000 dilution). After antibody binding, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Pierce Chemical). All incubations and washes were performed in PBS. Immunodetection was performed using the enhanced chemiluminescence method for western blotting detection (Pierce) followed by exposure to X-ray film. Band intensities were evaluated with the Image Station 440CF (Kodak, Rochester, NY). Mouse macrophage lysate prepared from the RAW 264.7 cell line (Transduction Laboratories, Lexington, KY) was used as a positive control for COX-2 expression.

Real-Time Quantitative Reverse Transcription-PCR

Total RNAs were prepared from pooled esophageal epithelia using TRIzol reagent (Invitrogen Corp., Carlsbad, CA). RNA samples were evaluated for integrity of 18S and 28S rRNA by agarose gel electrophoresis. Two micrograms of total RNA was reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Stratagene Corp., La Jolla, CA) at 42 °C for 1 hour using oligo d(T) (Promega Corp., Madison, WI). Triplicate samples of 20, 50, or 100 ng of cDNA were amplified by PCR in the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Unlabeled PCR primers Taqman MGB probe sets for COX-2 (LocusLink ID 29527), COX-1 (LocusLink ID 24693), and {beta}-actin (NM_031144) (Applied Biosystems: "Assay-on-Demand" gene expression products) were used to detect and quantify these sequences in RNA samples. The amplification reaction mixture (10 µL) contained cDNAs, primer–probe set, and Taqman Universal PCR Master Mix (Applied Biosystems). {beta}-Actin was amplified separately as an endogenous control to normalize for variable amounts of cDNA in each sample. Dilutions (range = 200 ng to 10 pg) of cDNA samples prepared from total ZS rat esophageal RNA were used to construct standard curves for the COX-2, COX-1, and {beta}-actin amplifications. The thermal cycling conditions were as follows: 50 °C for 2 minutes; 95 °C for 10 minutes; and 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. Relative quantitation with data obtained from AB 7900HT Sequence Detection System was performed using the standard curve method. Expression results for all samples were normalized to {beta}-actin. Normalized expression results for ZD and ZR esophagi were calculated relative to expression in ZS esophagi and expressed as fold-change above expression level of ZS esophagi.

Celecoxib and Indomethacin Treatment of ZD Rats

Twenty-eight weanling male Sprague–Dawley rats were fed a ZD diet and four rats a ZS diet for 5 weeks as described above. Twenty-four of the ZD rats were divided into three groups of eight. One group received an intragastric dose of celecoxib (CE) as a solution in PEG 400/saline (2 : 1, v/v) at 100 mg/kg body weight, another group received an intragastric dose of indomethacin (IM) as a solution of PEG 400/saline (2 : 1, v/v) at 40 mg/kg body weight, and the third group was treated with intragastric zinc replenishment as described above. As controls, four ZD and four ZS rats were treated intragastrically with the solvent PEG 400/saline and killed 8 hours later. Four rats from each drug-treated group and the ZR group were killed 4 hours after treatment and the other four were killed after 8 hours, forming CE4, CE8, IM4, IM8, ZR4 and ZR8 groups. Esophageal samples were isolated and prepared for immunohistochemistry as described above.

Immunohistochemical Detection of Cell Proliferation

Esophagus and tongue sections were deparaffinized and rehydrated in a graded alcohol series and then incubated with mouse anti–PCNA monoclonal antibodies (Santa Cruz) at a 1 : 250 dilution overnight at 37 °C in a humidified chamber, followed by incubation with biotinylated goat anti-mouse antibodies (DakoCytomation, Carpinteria, CA) at a 1 : 750 dilution and streptavidin horseradish peroxidase (DakoCytomation). PCNA staining was visualized by incubation with the chromogen 3-amino-9-ethylcarbazole (DakoCytomation). Cells whose nuclei showed strong staining for a red reaction product were defined as positive for PCNA, i.e., as being in S phase.

Immunohistochemical Analysis of COX-2, COX-1, Keratin 14, Bax, and Bcl-2 Protein Expression

Following deparaffinization and rehydration in a graded alcohol series, esophageal and lingual sections were heated in citrate buffer (0.01 M, pH 6.0) in a microwave oven (85–90 °C, 3 x 5 min) and nonspecific binding sites were then blocked with goat or rabbit serum. Sections were incubated overnight at 37 °C in a humidified chamber with one of the following primary antibodies: rabbit anti-COX-1 polyclonal antiserum (Santa Cruz) at a 1 : 60 dilution, rabbit anti-COX-2 polyclonal antiserum (Cayman Chemical) at a 1 : 50 dilution, mouse anti–keratin 14 monoclonal antiserum (Clone LL002, Novocastra, Newcastle upon Tyne, UK) at a 1 : 100 dilution, rabbit anti-Bcl-2 polyclonal antiserum (Santa Cruz) at a 1 : 400 dilution, or rabbit anti-Bax polyclonal antiserum (Abcam) at a 1 : 800 dilution (45 minutes; room temperature). All dilutions were in 0.5% bovine serum albumin in PBS. Sections were then incubated with the appropriate biotinylated secondary antibodies (DakoCytomation) and streptavidin horseradish peroxidase (DakoCytomation). Staining of individual proteins was visualized by incubating sections with 3,3'-diaminobenzidine tetrahydrochloride (DAB) and lightly counterstaining them with hematoxylin.

Apoptosis Detection

Apoptosis was detected in esophageal and lingual sections by the in situ oligo ligation (ISOL) assay, using an ApopTag ISOL assay kit (Serologicals, Norcross, GA). This assay relies on the selective binding of biotin-labeled hairpin oligonucleotide probes to the types of genomic DNA ends that are characteristic of the double-strand breaks in apoptotic cells. Briefly, the sections were deparaffinized, rehydrated in a graded alcohol series, and incubated with proteinase K at room temperature for 15 minutes. Endogenous peroxidase in the sections was inhibited with 3% hydrogen peroxide for 20 minutes. The slides were then incubated with T4 DNA ligase at 16–20 °C overnight as per the manufacturer’s instructions to catalyze blunt-end ligation of biotinylated oligo B with fragmented double-strand DNA. Slides were then incubated with streptavidin peroxidase conjugate, and oligo B binding (i.e., DNA fragmentation) was detected by staining with DAB, with methyl green as a counterstain. Sections from rat mammary gland, in which apoptosis is extensive, served as a positive control. Negative controls omitted T4 DNA ligase enzyme.

Tumor Induction by NQO

Thirty-seven weanling male Sprague–Dawley rats were fed a ZD diet ad libitum, and 11 additional rats were pair-fed a ZS diet to match the reduced food consumption of the ZD rats. All rats had free access to deionized water. After 5 weeks all 49 rats were switched to drinking water containing 5–15 ppm NQO for 15 weeks: 5 ppm for the first 6 weeks and 15 ppm NQO for the remaining 9 weeks. Average NQO intake was calculated by measuring the daily intake of water by five rats (per cage) over the entire experimental period and expressed as mg/day/kg body weight. The rats were observed daily for signs of ill health. To monitor the effect of ZD on NQO-induced carcinogenesis, 14 ZD rats were killed at week 6 (one rat), week 8 (three rats), week 9 (five rats), and week 10 (five rats) and 2 ZS rats were killed at week 6. For the analysis of tumor incidence the remaining rats were sacrificed at week 15. The rats were subjected to complete autopsies, with particular attention to tongue, esophagus, and forestomach. Tumors >0.5 mm in diameter were mapped and counted. Tongue, esophagus, and forestomach were fixed in phosphate-buffered formalin and embedded in paraffin; 4-µm sections were cut as described (13). Sections were either stained with hematoxylin and eosin (H&E) for histopathology or left unstained for immunohistochemical analysis.

Serum Zinc Analysis

At sacrifice, blood was collected from the retro-orbital venous plexus of each rat after anesthesia with isoflurane. The level of serum zinc was determined by atomic spectrometry using a Perkin–Elmer Atomic Absorption Spectrometer Analyst 100 (Perkin–Elmer, Norwalk, CT).

Statistical Analysis

Data on tumor multiplicity were analyzed by one-way analysis of variance (ANOVA), using the SAS statistical computer program (version 9.1, SAS, Cary, NC). Tumor incidence differences were analyzed by Fisher’s exact test, two-tailed. All statistical tests were two-sided and were considered statistically significant at P<.05.


    RESULTS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Serum Zinc Levels

We first determined the effect of the various zinc treatments on serum zinc levels (Table 1). Serum zinc levels in ZD rats (mean = 43 µg/100 mL) were approximately one-third those in control ZS rats (mean = 124 µg/100 mL; difference = 81 µg/100 mL, 95% confidence interval [CI] = 67 to 95). Two hours after zinc gluconate was administered to ZD rats, the level of serum zinc in ZR animals rose from a ZD level of 43 µg/100 mL to 605 µg/100 mL (difference = 562, 95% CI = 412 to 712). At 24 hours after ZR, the zinc level had decreased to 497 µg/100 mL (95% CI = 388 to 606), a result consistent with our previous report (30). Serum zinc levels in ZD rats treated with PEG 400/saline alone or PEG 400/saline containing celecoxib or indomethacin (data not shown) were similar to those in saline-treated ZD rats (Table 1). Serum zinc levels in the ZR4 and ZR8 rats used as controls in the COX-2 inhibition analyses (data not shown) were similar to those in the ZR2 and ZR8 rats in the zinc replenishment experiment (Table 1).


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Table 1. Serum zinc levels in experimental rats*

 
Lingual Cell Proliferation in ZD and ZR Rats

We next investigated whether ZD and ZR modulate cell proliferation in the rat tongue, as they do in the esophagus (13,18). We used PCNA immunostaining to identify cells in S phase (31). Tongues from pair-fed ZS control rats showed very mild proliferation; cells with PCNA-positive nuclei were largely restricted to the basal cell layers (Fig. 1, A and D) and found only occasionally in the suprabasal cell layer (Fig. 1, G). By contrast, ZD lingual epithelia displayed abundant PCNA-positive nuclei in many cell layers, including suprabasal layers (Fig. 1, B, E, H) and focal hyperplastic lesions (Fig. 1, E and H).



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Fig. 1. Zinc deficiency (ZD) and zinc replenishment (ZR) effects on lingual cell proliferation. Immunohistochemistry for proliferating cell nuclear antigen (PCNA) was used to identify proliferating cells. Representative pictures of tongue sections from individual rats are shown. A, D, G) In zinc-sufficient (ZS) control epithelia, PCNA-positive cells occurred mainly in the basal cell layer, with occasional staining in the suprabasal layer (arrow, G). B, E, H) In ZD epithelia, PCNA staining was abundant in many cell layers and focal hyperplastic lesions (arrows) (E, H). C, F, I) ZR epithelia had fewer cell layers and fewer PCNA-positive nuclei in the still-hyperplastic epithelium 8 hours after zinc replenishment (ZR8, C) and in focal hyperplastic lesions 12 hours after replenishment (ZR12, F). After 48 hours, ZR48 epithelium resembled that of ZS rats, with PCNA-positive nuclei largely restricted to the basal cell layer (I). Scale bars: 50 µm (in AC) and 25 µm (in DF).

 
ZR reduced cell proliferation, as assayed by PCNA staining, within hours. As early as 2 hours after an intragastric dose of zinc, ZR2 tongues showed reduced frequency of PCNA-positive nuclei in the still-proliferative epithelium (data not shown). At 8 and 12 hours, ZR8 (Fig. 1, C) and ZR12 (Fig. 1, F) lingual epithelia were thinner than those of ZD rats, with reduced numbers of PCNA-positive nuclei in suprabasal cell layers (Fig. 1, C) and in focal hyperplastic lesions (Fig. 1, F). By 48 hours PCNA-positive nuclei were mostly found in the basal cell layers of a thinned lingual epithelium (ZR48, Fig. 1, I).

COX-2 Protein Expression in Tongue and Esophagus of ZD and ZR Rats

To determine whether COX-2 protein expression is responsive to zinc manipulation in vivo, we performed immunoblot analysis. COX-2 protein was expressed at low levels in ZS tongue and esophagus, a result consistent with COX-2 expression in normal cells (20), whereas COX-2 expression was strongly induced during zinc deprivation (Fig. 2, A and B). Relative to expression in ZS controls, COX-2 expression in ZD tongue was increased by 11-fold and 8-fold (in two separate pooled samples; Fig. 2, E), and in ZD esophagus by almost approximately 12-fold (Fig. 2, F). Within hours, a single intragastric zinc treatment restored COX-2 expression to near normal levels in both tongue and esophagus of ZR rats. For example, at 8 and 12 hours, COX-2 levels in tongues of ZR rats were approximately fourfold and twofold higher, respectively, than those in ZS tongues, and by 48 hours COX-2 levels had returned to almost that of ZS tongue (Fig. 2, E). Similar results were seen in ZR esophagi (Fig. 2, F). Expression of COX-1 protein in esophagus and tongue was, by contrast, not affected by zinc (Fig. 2, C and D).



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Fig. 2. COX-1 and COX-2 protein expression in tongue and esophagus from zinc-modulated rats. Representative immunoblots are shown. Each band represents a separate sample, pooled from three to five esophagi or two tongues. Positive control for COX-2 was derived from mouse macrophage lysate. A, B) COX-2 protein expression in tongue (A) and esophagus (B). C, D) COX-1 protein expression in tongue (C) and esophagus (D). E, F) Folds above ZS were calculated from the ratio of COX-2 to GAPDH levels in tongue (E) and esophagus (F). ZS = zinc-sufficient control, ZD = zinc-deficient, and ZR2, ZR4, ZR8, ZR12, ZR24, ZR48 represent 2, 4, 8, 12, 24, and 48 hours after zinc replenishment.

 
Temporal and Spatial Localization of COX-2 Protein in ZD Esophagus and Tongue

To confirm and expand on the results obtained by immunoblotting, COX-2 protein expression was analyzed in esophageal and lingual sections by immunohistochemistry. Invariably, ZS esophageal epithelial cells showed weak and diffuse cytoplasmic staining of COX-2 (Fig. 3, A). By contrast, ZD esophagi exhibited frequent and strong to intense COX-2 staining in the highly proliferative epithelium (Fig. 3, B–F). In addition, COX-2 overexpression in ZD esophageal epithelia displayed notable spatial and temporal patterns. For example, intense COX-2 staining was observed in many cell layers throughout an entire esophageal section (Fig. 3, B), with characteristic perinuclear cytoplasmic staining (Fig. 3, C); in bands that alternated with zones of no expression in a highly proliferative section (Fig. 3, D); in parakeratotic regions of the outermost cell layers (Fig. 3, F); and in the hyperplastic cell layers but not in parakeratotic regions of the outer layers (Fig. 3, E). Within hours of intragastric zinc administration COX-2 staining in esophagus of ZR rats showed a substantial reduction in staining intensity and number of cells stained. For example, COX-2 staining in a ZR8 esophageal section still occurred in bands resembling those in ZD rats, but with reduced intensity (Fig. 3, G versus D). By 48 hours, the esophageal epithelium was mostly thinned (18), and COX-2 expression was mostly diffuse and weak (data not shown).



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Fig. 3. Spatial and temporal localization of cyclooxygenase-2 (COX-2) protein in esophagus and tongue from zinc-modulated and COX-2 inibitor-treated rats. Representative immunohistochemically stained sections are shown. AI) Esophagus. JO) Tongue. A) COX-2 staining was mostly absent in zinc-sufficient control esophagi. BF) COX-2 was intensely and abundantly expressed in zinc-deficient (ZD) esophagi, occurring in different spatial and temporal patterns (arrows) and with typical perinuclear cytoplasmic staining (C, arrows). GI) COX-2 expression was reduced within hours after zinc replenishment (ZR) or treatment with celecoxib (CD) or indomethacin (IM). ZR8 esophagi (G) showed weaker staining in patterns similar to those in ZD esophagi (compare with D). CE8 (H) esophagi showed reduced COX-2 staining compared with ZD esophagi (compare with H and D) but stronger overall staining than ZR8 (compare with G). IM8 (I) also showed reduced COX-2 staining compared with ZD esophagi but stronger overall staining than ZR8. J) ZS tongue showed only sporadic COX-2 expression, whereas ZD tongue displayed intense and abundant COX-2 expression (KM), covering many cell layers (K and L) and in outer cell layers (M). NO) COX-2 staining was reduced in intensity and scope, as shown in sections from ZR12 (N) and ZR48 (O) rats. ZR8, ZR12, and ZR48 represent ZR rats at 8, 12, and 48 hours after replenishment. CE8 and IM8 represent inhibitor-treated rats at 8 hours after treatment. Scale bars: 100 µm (in A, B, J, K); 50 µm (in DI and LM); 25 µm (in C and in insets of J, K, M, N).

 
In ZS tongues, COX-2 staining was mostly absent or very weak, with infrequent strong cytoplasmic staining in a few suprabasal cells (Fig. 3, J). As in the esophagus, an increase in intensity of COX-2 staining and number of COX-2-stained cells was evident in lingual epithelia (Fig. 3, K–M), which exhibited spatial patterns of COX-2 expression similar to those in ZD esophagi (Fig. 3, B–F). Again, within hours after zinc administration, COX-2 staining in the lingual epithelium of ZR rats decreased substantially (Fig. 3, N and O). For example, at 8 hours, COX-2 staining was moderately strong in many cells in the still-proliferative lingual epithelium (Fig. 3, N). By 48 hours, COX-2 staining was found in only a few cells (Fig. 3, O). Moderate cytoplasmic COX-1 staining was detected in ZS tongue and esophagus (data not shown). There was no marked difference in staining pattern or intensity of COX-1 staining in ZD and ZR samples as compared with ZS (data not shown).

COX-2 mRNA Expression in ZD and ZR Esophagus

We next investigated whether COX-2 mRNA would respond to zinc in a manner similar to COX-2 protein. Real-time quantitative RT-PCR was used to assess COX-2 mRNA expression in ZS and ZD esophagi and in ZR esophagi at 8, 12, and 48 hours after zinc administration (Table 2). The level of COX-2 mRNA was low in normal ZS esophagus (mean = 14 ng total esophageal RNA) but was greatly elevated in ZD esophagus (mean = 247 ng total esophageal RNA, difference = 233 ng, 95% CI = 201 to 265). {beta}-Actin was used to normalize variations in cDNA quantities. As Table 2 shows, {beta}-actin mRNA levels were 20% higher in ZD esophagus than ZS esophagus, a result that is consistent with a reported 15% increase in actin mRNA expression in small intestine during zinc deficiency (32). However, because the change in {beta}-actin mRNA levels is minor compared with that of COX-2, normalizing to actin is appropriate (Table 2). Thus, after normalization to {beta}-actin, ZD esophagus showed a 14.7-fold increase in COX-2 mRNA expression relative to the level in ZS esophagus. After ZD rats were administered intragastric zinc, levels of COX-2 mRNA dropped rapidly. Within 8 hours, COX-2 mRNA levels were reduced to approximately threefold those in ZS esophagus (Table 2). By contrast, COX-1 mRNA expression was not changed in ZD and ZR rats.


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Table 2. Real-time quantitative polymerase chain reaction assessment of cyclooxegenase-1 (COX-1) and COX-2 mRNA expression in rat esophagus*

 
COX-2 Expression, Cell Proliferation, and Marker Gene Expression in Esophagi of ZD Rats Treated with COX-2 Inhibitors

To determine whether cell proliferation and apoptosis in esophagi of ZD rats administered intragastric zinc (18) was mediated by altered COX-2 levels, we analyzed cell proliferation and apoptosis in ZD rats treated with COX-2 inhibitors. A single intragastric dose of celecoxib (a selective COX-2 inhibitor) or indomethacin (an inhibitor of both COX-1 and COX-2) was administered to ZD rats, and the rats were killed 4 and 8 hours after treatment. Eight hours after treatment with celecoxib or indomethacin, COX-2 overexpression in ZD esophagi was reduced (Fig. 3, H and I), although to a lesser extent than it was reduced by zinc administration (compare Fig. 3, G). A reduction in cell proliferation, as assayed by PCNA staining, and an increase in apoptosis were evident in the ZR and celecoxib- and indomethacin-treated esophagi as early as 4 hours after treatment (data not shown). However, at 8 hours, fewer focal hyperplastic lesions were present in the ZR esophagi than in esophagi from drug-treated ZD rats (data not shown).

Consistent with previous findings (15,16,18), ZD esophagi typically showed PCNA-positive nuclei in several cell layers and in focal hyperplastic lesions (Fig. 4, A), strong cytoplasmic staining for the esophageal tumor marker keratin 14 (16) in the proliferative cell layers and in focal hyperplastic lesions (Fig. 4, E), sporadic occurrence of apoptotic nuclei (Fig. 4, I), diffuse and weak staining for the proapoptotic protein Bax (Fig. 4, M), and strong cytoplasmic staining for the anti-apoptotic protein Bcl-2 (Fig. 4, Q). In the ZR and inhibitor-treated ZD rats, at 8 hours after treatment, PCNA-positive nuclei were found mostly in the basal cell layers in the still-proliferating esophagi from ZR (Fig. 4, B), celecoxib- (Fig. 4, C), and indomethacin-treated (Fig. 4, D) rats. Keratin 14 overexpression in the ZD esophagi (Fig. 4, E) was reduced by all three treatments (Fig. 4, F–H), although the reduction was greater in ZR rats than in celecoxib- or indomethacin-treated rats (Fig. 4, F versus G and H). All three treatments induced a wave of apoptosis (Fig. 4, J–L) and overexpression of Bax (Fig. 4, N–P). Apoptotic nuclei were more numerous in esophagi from ZR or celeboxib-treated rats than in esophagi from indomethacin-treated rats (Fig. 4, J and K versus L). In agreement with these results, esophagi from ZR and celecoxib-treated rats showed stronger Bax expression (Fig. 4, N and O versus P) and weaker Bcl-2 staining (Fig. 4, R and S versus T) than did esophagi from indomethacin-treated rats.



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Fig. 4. Protein expression and apoptosis in esophagi of zinc-modulated and COX-2 inhibitor–treated zinc-deficient (ZD) rats. Zinc-replenished (ZR) and inhibitor-treated rats were killed 8 hours after treatment. Representative sections, stained as described in "Methods," are shown. AD) Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), an internal marker for cell proliferation, showed many PCNA-positive nuclei in the basal/suprabasal cell layers and focal hyperplastic lesions (FHLs) of ZD esophageal epithelia (A) but few such cells, mainly in the basal cell layer, of the esophagi of ZR rats (B) or rats treated with celecoxib (CE) (C) or indomethacin (IM) (D). EH) Immunohistochemical staining for the esophageal tumor marker keratin 14 showed strong expression in the cytoplasm of the basal cell layers and in FHLs of ZD esophagi. (E), weak expression in the cytoplasm of the basal cell layers of ZR (F) and IM (H) rats, and moderate expression in CE rats (G). IL) In situ oligo ligation analysis of apoptosis showed occasional apoptotic nuclei in ZD esophagus (I) but numerous apoptotic bodies (arrows) in ZR (J), CE (K), and IM (L) esophagi. MP) Immunohistochemical staining for the proapoptotic protein Bax showed weak expression in the cytoplasm of ZD esophagi showing hyperplasia (M), strongly expression in suprabasal cells of ZR (N) and CE (O) esophagi, and moderate expression in the basal cells of IM esophagi (P). QT) Immunohistochemical staining for the anti-apoptosis protein Bcl-2 showed strong expression in the cytoplasm of basal cells and focal hyperplastic lesions in ZD esophagi showing hyperplasia (Q), weak and diffuse expression in suprabasal cells of ZR (R) and a CE (S) esophagi, and moderate expression in the basal cells of an IM esophagus (T). Scale bars: 50 µm (in AH); 25 µm (in IT).

 
NQO Induction of Tumors in ZD Rats

We next examined the induction of tumors by the carcinogen NQO, which is known to cause tongue abnormalities in nutritionally complete rats (29). To ensure that carcinogen uptake was similar in the two kinds of rats, we measured the daily intake of water by five rats (per cage) and calculated the quantity of NQO intake per day per kilogram of body weight. Average intakes were similar during weeks 1–6, when drinking water contained 5 ppm NQO (0.48 mg/day/kg body weight in ZD rats and 0.49 mg/day/kg body weight in ZS rats, difference = 0.01 mg, 95% CI = 0.02 to 0.04), and during weeks 7–15, when drinking water contained 15 ppm NQO (0.92 mg/day/kg in ZD rats and 1.00 mg/day/kg in ZS rats, difference = 0.08 mg, 95% CI = 0.06 to 0.09). Because ZS rats were pair-fed to ZD animals to match their reduced food consumption, their body weights were similar throughout the NQO feeding experiment and at week 15 (ZD = 247 g, ZS = 267 g, difference = 20 g, 95% CI = 13 to 53). As expected, the level of serum zinc at week 15 was statistically significantly lower in ZD rats (42 µg/100 mL) than in ZS rats (150 µg/100 mL, difference = 108 µg/100 mL, 95% CI = 97 to 119, P<.001).

To monitor the influence of ZD on the course of lingual carcinogenesis, 14 ZD rats were killed at random times between 6 and 10 weeks after the start of NQO treatment. At week 6, one ZD rat showed three leukoplakia lesions in the anterior dorsum of the tongue, two small papillomas in the proliferative esophagus, and fused tumors in the squamocolumnar junction (SCJ) between the forestomach and the glandular stomach. After week 8, increases were evident in lingual tumor burden and tumor size in ZD rats and in the occurrence of esophageal and forestomach tumors (data not shown). Two ZS rats killed at week 6 displayed a normal tongue, esophagus, and forestomach.

At week 15, all NQO-treated ZS and ZD rats had lingual tumors (Table 3). The ZD rats, however, showed statistically significantly greater tumor burden and tumor sizes than the ZS rats and had a higher incidence of squamous cell carcinomas. Mean tumor multiplicity was 13.1 in the ZD rats but only 4.3 in the ZS rats (difference = 8.8, 95% CI = 7.0 to 10.6, P = .018). NQO-treated ZD rats also had larger tumors that often covered the entire dorsum (Fig. 5, A). Twenty of 23 (87%) ZD rats versus 2 of 9 (22%) ZS rats had tumors larger than 2 mm (P = .001). In addition, the incidence of lingual SCC was statistically significantly greater in the ZD rats than in the ZS rats (74% [17 of 23 ZD rats] versus 22% [2 of 9 ZS rats]; P = .015). Examples of lingual SCC from two ZD rat are shown in Fig. 5, D and G. Dysplasia was seen frequently in the tongues of both ZD (data not shown) and ZS rats (Fig. 5, J).


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Table 3. Incidence of lingual, esophageal, and forestomach tumors in rats after 15 weeks of treatment with 4-nitroquinoline 1-oxide (NQO)*

 


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Fig. 5. Characterization of 4-Nitroquinoline 1-oxide (NQO)-induced lingual, esophageal, and forestomach tumors. AC) Macroscopic appearance and histopathology of the tongue, esophagus, and forestomach of zinc-deficient (ZD) rats. Rat 32 showed numerous tumors on dorsal and lateral tongue (A), rat 34 had a large tumor in the lower esophagus and three small tumors in the upper and lower esophagus (B), and rat 36 had large coalesced tumors in squamocolumnar junction between forestomach and glandular stomach (SCJ) and forestomach (C). DL) Hematoxylin and eosin–stained sections. NQO-treated ZD rats displayed frequent occurrence of tongue carcinomas (D, G) and forestomach carcinomas (F, I). They also harbored esophageal carcinomas (E) and esophageal glandular metaplasia (H, inset showing higher magnification). NQO-treated zinc-sufficient (ZS) control rats typically showed hyperplastic tongue with occasional dysplasia (J) and esophagus (K) and forestomach (L) with mild hyperplasia. MO) COX-2 staining was moderate in an area of NQO-treated ZS tongue with hyperplastic cell layers (M) and strong in invasive squamous cell carcinomas of ZD tongue (N) and in ZD esophageal squamous cell carcinomas. Scale bars: 5 mm (in AC); 200 µm (in H, O); 100 µm (in DF, I, L); 50 µm (in G, J, K, M, N); and 25 µm (in inset of H).

 
NQO-treated nutritionally complete rats are not known to develop macroscopic abnormalities in organs other than the tongue (29). However, 9 of 23 (39%) NQO-treated ZD rats also harbored esophageal tumors (Fig. 5, B), and 14 of 23 (61%) had forestomach tumors (Fig. 5, C), as compared with none of the NQO-treated ZS rats. The difference was statistically significant at P = .034 for esophagus and at P = .002 for forestomach. In addition, among the 23 ZD rats, glandular esophageal metaplasia was detected in 1 rat (4%; Fig. 5, H); invasive ESCC in 1 rat (4%; Fig. 5, E), and invasive forestomach SCC in 5 rats (22%; Fig. 5, F and I). By contrast, esophagi (Fig. 5, K) and forestomach (Fig. 5, L) from NQO-treated ZS rats were tumor-free and displayed hyperplasia only.

COX-2 Overexpression in Lingual and Esophageal SSC from NQO-Treated ZD Rats

We investigated whether the tumors induced by NQO in ZD rats were accompanied by COX-2 overexpression and found that lingual (Fig. 5, N), esophageal (Fig. 5, O), and forestomach (data not shown) squamous cell carcinomas from NQO-treated ZD rats typically displayed strong COX-2 staining in the tumor areas. By contrast, NQO-treated ZS rats at endpoint showed moderate COX-2 staining in the basal cells in hyperplasia in lingual epithelia (Fig. 5, M), and moderate to strong COX-2 expression in tumor areas (data not shown), a result consistent with the findings of Shiotani et al. in similarly treated nutritionally complete rats (29).


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study shows that exposing rats to ZD conditions increased the expression of COX-2 protein and mRNA in the esophagus and tongue by as much as 14.7-fold, as compared with the ZS controls. An intragastric dose of zinc or treatment with COX-2 inhibitors celecoxib or indomethacin rapidly and effectively reduced COX-2 overexpression and reversed the hyperplastic phenotype of the esophagus of ZD rats (18) (Figs.24; Table 2). A similar result was observed in the tongue with zinc replenishment. By contrast, ZD and ZR had no effect on the expression of COX-1. In addition, the results show that COX-2 overexpression is accompanied by a hyperplastic phenotype in ZD esophagus and tongue that are likely relevant to cancer development (33). The reversal of the phenotypic changes that accompany ZD by two different treatments that reduce COX-2 activity (ZR or treatment with inhibitors) support a direct role for overexpression of COX-2 in the increased susceptibility to esophageal carcinogenesis seen in ZD rats. Such a conclusion is supported by the known roles of COX-2 in carcinogenesis. COX-2 overexpression leads to an increased production of prostaglandins, including prostaglandin E2, which stimulates epithelial cell proliferation and inhibits apoptosis (46). Our previous work (18) showed that ZR rapidly stimulates apoptosis by increasing Bax expression, thereby arresting esophageal carcinogenesis in rats induced by NMBA. ZR also stimulates Bax expression in rat tongues (Fong and Farber, unpublished results).

Zinc is an essential nutrient that is required as a cofactor for the activity of more than 300 enzymes in mammals. Zinc is also a component of many additional proteins, mainly nuclear transcription factors that regulate proteins involved in cell proliferation, differentiation, and apoptosis (34). Thus, alterations in zinc levels are expected to translate into changes in gene expression. Blanchard et al. (32) first reported that dietary ZD modulates intestinal gene expression in vivo: expression of 16 genes that influence signaling pathways, growth, redox, and energy utilization was increased in ZD rats, and expression of 16 other genes of this type was decreased. Recently, we performed oligonucleotide array analysis using same RNA samples as were used in the current study to compare the expression profiles of genes in ZD, ZS, and ZR esophagi at 8–48 hours after the ZD rats were dosed with intragastric zinc (35). The biochip used did not contain the COX-1 or COX-2 gene. A total of 33 genes changed in expression level by more than twofold (expression of 21 genes was increased and of 12 was decreased) in ZD versus ZS esophagus. Levels of all 33 genes returned to near normal levels (i.e., levels in ZS esophagus) 48 hours after ZR. For example, levels of keratin 14, a biomarker of esophageal carcinogenesis (16,36), were sixfold higher in ZD esophagus than in ZS esophagus but returned to nearly normal levels 48 hours after ZR. The present study used immunoblotting, immunohistochemistry and real-time quantitative PCR to identify zinc as a modulator of COX-2 expression. Together, the data from these analyses show that certain genes in the rat esophagus and tongue are highly responsive to ZD and ZR, and likely, therefore to be relevant to esophageal and oral carcinogenesis. These genes are induced during rapid transition from zinc deficiency to sufficiency by ZR and may be responsible for a reversal of the precancerous environment in the ZD esophagus and tongue. Thus, it is not surprising that the COX-2 inhibitors are less effective than ZR in reversing the numerous genetic abnormalities in the esophagus caused by ZD (Fig. 4).

Additional nutrients have been reported to affect COX-2 expression in vitro. For example, selenium supplementation selectively reduced COX-2 expression in activated RAW 264.7 macrophages (37). Vitamin C reduced COX-2 overexpression mediated by ethanol in glial cell (38), and retinoids suppressed phorbol ester-mediated induction of COX-2 in human oral epithelial cells (39). However, our study is the first, to our knowledge, to demonstrate in vivo that a deficit of an essential nutrient, zinc, is associated with COX-2 overexpression and a phenotype with increased tumorigenic potential and that zinc treatment corrects COX-2 overexpression and the tumorigenic phenotype.

Here, we found that continuous exposure of ZD rats to NQO resulted in a high incidence of lingual SCC (74%), esophageal (39%), and forestomach tumors (61%), whereas ZS rats continuously exposed to NQO showed no macroscopic abnormalities in the esophagus or the forestomach (Table 3) (29). We believe that the development of second primary tumors in NQO-treated ZD rats is a manifestation of "field cancerization," a theory first put forward by Slaugher et al. (4) that the entire UADT is at increased risk for the development of malignant lesions when there are multiple genetic abnormalities in the whole region. Our data provide what we believe to be the first evidence that ZD causes substantial cell proliferation in the squamous epithelium of the UADT, thereby providing a fertile environment for the genetic events that culminate in the development of malignant lesions at multiple sites after continuous exposure to NQO. Our ZD rat tongue cancer model mimics aspects of human UADT cancer. Patients with UADT cancer are frequently zinc-deficient (10,11), and they often present with second primary cancers at diagnosis (46). This ZD oral cancer model will be useful to identify novel molecular targets in UADT carcinogenesis and to test candidate chemoprevention agents and combination therapeutic strategies. The relevance of our rat model to human UADT cancers will depend, of course, on epidemiologic studies that further define the relationship between environmental zinc deficiency and the occurrence of these tumors.

ZD is more widespread than is often assumed. Although zinc is present in a large variety of foods, its levels are low in most foods except for meat and seafood. Thus, a person subsisting on a cereal diet is likely to have low zinc intake. It has been estimated that approximately 10% of the US population ingests less than 50% of the recommended daily allowance of zinc (40), and dietary zinc deficiency may affect more than 2 billion people in the developing world (41). Importantly, ZD is implicated in the etiologies of esophageal and oral cancer in humans. In this study, we have shown that zinc treatment successfully and efficiently corrects COX-2 overexpression induced by ZD, thus providing preclinical evidence to support the use of zinc as a potential COX-2 inhibitor in UADT tissues. High doses of retinoids have established efficacy in reversing oral leukoplakia (early premalignant lesions), but this treatment is associated with mucocutaneous toxicity (42). It may be possible to use zinc in combination with lower doses of retinoids and selective COX-2 inhibitors, such as celecoxib, to reduce their toxicity. More broadly, our study raises the possibility that zinc supplementation may have a role in the prevention of oral and esophageal cancer in persons who are at high risk for the disease because they have a low dietary intake of this nutrient.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by grant # 02A025-REV (LYYF) from the American Institute for Cancer Research.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received May 18, 2004; revised August 10, 2004; accepted November 1, 2004.


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