Over-expression of gastrin-releasing peptide in human esophageal squamous cell carcinomas

Ming Zhu Fang1, Changgong Liu1, Yunlong Song1, Guang-Yu Yang1, Yan Nie1, Jie Liao1, Xin Zhao2, Yutaka Shimada3, Li-Dong Wang2 and Chung S. Yang1,4

1 Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA, 2 Laboratory for Cancer Research, College of Medicine, Zhengzhou University, Zhengzhou, Henan 450052, China and 3 Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan

4 To whom correspondence should be addressed. Email: csyang{at}rci.rutgers.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Gastrin-releasing peptide (GRP) is known as an autocrine growth factor for a number of gastrointestinal cancers. There is, however, little information on the expression of GRP in the squamous epithelia and squamous cell carcinoma, particularly in the esophagus. With a differential display approach, up-regulated GRP was observed in human esophageal squamous cell carcinoma (ESCC) samples obtained from a high-risk area for esophageal cancer, Linzhou in northern China. Up-regulation of phosphoglycerate mutase and P311 HUM (3.1) and down-regulation of keratin 13, cystatin B, endoglin and annexin I were observed. Using a reverse transcription-polymerase chain reaction (RT–PCR) method, significant over-expression of GRP was observed in 10 out of 12 ESCC samples (83.3%) and all four ESCC cell lines. With in situ hybridization, GRP mRNA expression was detected in nine out of 21 (42.8%) samples with basal cell hyperplasia (BCH), five out of seven (71.4%) samples with dysplasia (DYS) and 17 out of 24 (70.9%) ESCC samples. In contrast, GRP was expressed only in three out of 16 (18.7%) normal epithelium. Digital image analysis revealed that the mean value of GRP expression index, determined by intensity and area ratio of staining, was 0.19 in normal epithelium, 1.23 in BCH, 2.94 in DYS and 2.38 in ESCC, showing a progressive increase. Studies on ESCC cell lines showed GRP increased cell growth in a dose-dependent pattern in GRP receptor-positive ESCC cells, but not in GRP receptor-negative ESCC cells. GRP (1 mM) also increased cyclooxygenase-2 protein expression by 3.4-fold. This is the first demonstration that GRP is over-expressed in ESCC, and its over-expression may play a role in ESCC development and growth.

Abbreviations: BCH, basal cell hyperplasia; Cox-2, cyclooxygenase 2; DIG, digoxigenin; DYS, dysplasia; ESCC, esophageal squamous cell carcinoma; EST, Expressed Sequence Tags; GRP, gastrin-releasing peptide; GRPR, gastrin-releasing peptide receptor; RT–PCR, reverse transcription–polymerase chain reaction


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Human esophageal cancer is the sixth most common cancer in the world, of which esophageal squamous cell carcinoma (ESCC) accounts for >90% of the cases. Epidemiological studies have identified the use of tobacco and consumption of alcohol as major risk factors for ESCC (1). Nitrosamines, mycotoxins, physical injury and chronic inflammation have also been suggested to be possible etiological factors (2). With similar life styles, environment and genetic backgrounds, the specimens obtained from the high-risk area, Linzhou (formerly named Linxian) in Henan, China provided a good opportunity to study its molecular alterations in the development of ESCC. Our previous studies suggest that inactivation of p53 and Rb tumor suppressor systems are frequent events in esophageal carcinogenesis (3,4), and p53 mutation is likely to occur in the early stage (5,6). Loss of heterozygosity and microsatellite instability of the tumor suppressor gene cluster 9p21 (p14ARF, p15INK4b and p16INK4a) and hypermethylation of p16INK4a, p14AFR and HLA class I genes are also frequent early events in ESCC carcinogenesis (79). High levels of epidermal growth factor receptor have also been observed in some ESCC cases (10). The involvement of the ras and myc genes in ESCC have been studied extensively, but point mutations of the H-, K- or N-ras genes and the amplification of the c-myc gene have rarely been detected in samples from different high-risk areas (1114). Recently, dozens of up-regulated or down-regulated genes have been observed in ESCC with cDNA microarray, of which oncogenes Fra-1 and Neogenin are over-expressed in ESCC (1517). However, the major growth promoting factors in esophageal squamous carcinogenesis are not known. In this study, we used a differential display approach to study up-regulated and down-regulated genes to explore novel mechanisms that may be involved in human esophageal carcinogenesis; of the several up-regulated genes, gastrin-releasing peptide (GRP) was selected for detailed investigation.

GRP, a 27 aa small peptide, is the mammalian homolog of amphibian tetradecapeptide, bombesin (18). Human GRP has been isolated, and the gene has been localized to chromosome 18p26 (19). GRP acts as a neurotransmitter in the brain, as a paracrine hormone in the gastrointestinal tract and as a growth factor in the developing lung (20). In the mammalian gastrointestinal tract, GRP induces secretion of hormones, gastric acid and mucin; regulates smooth muscle contractions; and modulates neuronal firing rate (21). High levels of GRP immunoreactivity occur in the human fundus, antrum, pylorus and pancreas, whereas lower levels occur in the duodenum, jejunum, terminal ileum and colon (22). GRP acts by binding to the GRP receptor (GRPR), a specific G-protein coupled receptor with seven transmembrane spans (23). GRPR is widely expressed in the central and enteric nervous systems, where they act to alter a number of normal physiological processes including satiety, thermoregulation, circadian rhythm, smooth muscle contraction, immune function and the release of other peptide hormones (24). It is also distributed in the epithelium of the gastric antrum, but not in esophageal squamous epithelium (23,25).

GRP over-expression has been reported in lung, breast, renal, prostate, thyroid, stomach, intestine and colon cancers and malignant melanoma (24,26). There is, however, little information on the expression of GRP in the squamous epithelia and squamous cell carcinoma, particularly in the esophagus. Recent studies have shown that GRPR are aberrantly expressed in human gastrointestinal, ovarian and prostate cancers (25,2729). Nevertheless, the GRPR expression in esophageal cancer is not clear.

Evidence from studies on small cell lung cancer and non-small cell lung cancer indicates that GRP stimulates growth of cells in vivo and in vitro in either autocrine or paracrine fashions (3032). GRP is also a potential mitogen for Swiss 3T3 mouse fibroblast and for N-ras transfected NIH 3T3 cells (33). Millar and Rozengurt reported that arachidonic acid release along with prostaglandin E2 formation is an early signal in the mitogenic response to GRP (34). Recently, it was reported that GRP induced activation protein 1 (AP-1) gene expression and binding activity (35). GRP-induced AP-1 transcription factor also mediated cyclooxygenase-2 (Cox-2) expression in intestinal epithelial cells (36). Monoclonal antibodies against GRP or GRPR antagonists could inhibit GRP functions (3739). Therefore, GRP was suggested as a possible tumor therapy target (37).

In this report, we demonstrated the over-expression of GRP mRNA in ESCC with differential display and confirmed the over-expression with RT–PCR. Studies with in situ hybridization indicated the progressive increase of GRP expression in esophageal squamous carcinogenesis. In GRPR-positive ESCC cells, GRP increased cell proliferation and Cox-2 expression.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Specimen preparation
Primary ESCC specimens containing neighboring normal epithelial tissues were collected from patients in Linzhou People's Hospital, Linzhou, Henan, China. The samples were frozen in liquid nitrogen within 1 h after surgical resection, and were stored in liquid nitrogen, dry ice or in –80°C freezers before use. For each specimen, several pieces of tissues, some from the tumor mass and others from normal esophageal mucosa, were dissected and embedded with tissue freeze medium (OTC). Normal, basal cell hyperplasia (BCH), dysplasia (DYS) or ESCC samples were diagnosed histopathologically on slides stained with hematoxylin and eosin (H&E) by two pathologists (G.-Y.Y. and J.L.).

Total RNA was extracted from ESCC tissues and adjacent non-tumorous tissues using the RNA Mini kit (Qiagen, Valencia, CA) following the manufacturer's instruction, and then stored at –80°C. Frozen embedded freezing tissues were cryosectioned to a thickness of 10 µm and placed on RNAse-free slides. After air-drying, these slides were stored at –80°C before in situ hybridization.

ESCC cell lines, KYSE 150, 190, 450 and 510 (40), were maintained in RPMI 1640 and Ham F12 mixed (1:1) medium containing 5% fetal bovine serum. Total RNA was also extracted using the RNA Mini kit after cells were cultured to 80% confluence. Cells were seeded in 8-well slide chambers at 1 x 104 cells/well using the above medium. After reaching 80% confluence, cells were fixed in 4% paraformaldehyde and permeabilized in 0.3% Triton X-100. After being hydrated in gradient alcohol, the cells were air-dried and refrigerated before being analyzed by in situ hybridization.

Differential display
Total RNA prepared from two pairs of ESCC and adjacent non-tumorous epithelial samples were used to synthesize the first strand cDNA with an AdvantageTM RT-for-PCR kit (Clontech, Palo Alto, CA) following the protocol provided in the kit. Twenty-four PCR reactions were conducted with three anchor primers combined with eight arbitrary primers (GenHunter, Nashville, TN). PCR products were resolved on 6% polyacryamide gel. Differentially expressed bands were cut out, purified using the Qiagen Gel Extraction kit, and re-amplified with corresponding differential display primers. Re-amplified PCR products were sequenced using the Automatic Sequencer, maintained and conducted by the DNA Core Facility of the UMDNJ-Robert Wood Johnson Medical School. The Gene Bank Blast program was used for identification of the gene sequence.

RT–PCR
Reverse transcription was performed using the AdvantageTM RT-for-PCR kit. PCR primers for GRP, whole length GRPR and ß-actin control were designed by Primer 3 software, developed and maintained by the Whitehead Institute/MIT Center for Genomic Research (Table I). In order to produce a gene-specific mRNA signal, the total RNA was treated with DNase I to remove genomic DNA contamination. Diethyl pyrocarbonate-treated water was used as a negative control and total RNA from human small cell lung cancer tissue (Novagen, Madison, WI) was used as positive control for GRP mRNA expression. PCR cycle numbers (typically 27–30 cycles) were determined to keep the reaction in the linear stage. PCR products were run in 2% agarose gel and confirmed by sequencing using an Automatic Sequencer. Signal intensities were quantified using densitometry (Bio-Rad, Hercules, CA). The level of GRP mRNA was quantified by the intensity ratio of the target signal to the ß-actin control under the same PCR reaction conditions. We used the cut-off of (density of GRP/density of actin)tumor/(density of GRP/density of actin)normal >10 to distinguish significant over-expression.


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Table I. RT–PCR primer sequences

 
In situ hybridization
A non-radioactive in situ hybridization protocol was adapted for the determination of GRP mRNA expression (41,42). In brief, GRP cDNA was produced by RT–PCR using the total RNA from human small cell lung cancer as a template and ligated into pCR II-TOPO vector, then transformed into competent E.coli using the TOPO TA cloning kit from Invitrogen (Carlsbad, CA). The cloned human GRP cDNA was amplified, purified and linearized with HindIII or NotI for antisense or sense cRNA probe. Digoxigenin (DIG)-labeled antisense and sense cRNA probes were prepared using a DIG T7/SP6 transcription kit (Boehringer-Mannheim, Indianapolis, IN) from linearized plasmid human GRP cDNA. The quality and specificity of the probe were determined on the positive control slides made from the human pancreas (Novagen) (data not shown). The sense probe or probe vehicle was used as a negative control to verify the specificity of the antisense cRNA probe.

The sliced tissue was fixed in 4% paraformaldehyde, digested with proteinase K and then acetylated in freshly prepared 0.25% acetic anhydride in a 0.1 M triethanolamine buffer. Hybridization buffer (50% deionized formamide, 25 mM phosphate buffer, pH 7.4, 2x standard saline citrate, 2x Denhardt's solution, 10% dextran sulfate, 0.02 M dithiothreitol, 0.4 mg/ml yeast tRNA, 250 µg/ml salmon-sperm DNA in diethylpyrocarbonate-treated water) containing 400 ng/ml of probe was placed onto the permeabilized section and covered with a paraffin coverslip. Then the slides were denaturated at 80°C and hybridized at 42°C overnight. After post-hybridization washing in 1x SSC containing 2% normal sheep serum and 0.05% Triton X-100 for 2 h, the slides were blocked for 30 min in 0.1 M maleic acid buffer (pH 7.5) containing 2% normal ship serum and 0.3% Triton X-100, and then incubated for 1 h with a sheep anti-DIG antibody (1:1000) and in streptavidin-alkaline phosphatase conjugate for 20 min. Finally, the color was developed in a NBT/BCIP chromogen solution. Between each reaction, the slides were washed three times for 5 min each in 0.1 M maleic acid buffer (pH 7.5) containing 0.1% Triton X-100. After being washed in tap water, the slides were then mounted with Aqua-mounting medium (Fisher, Houston, TX).

The stained sections were reviewed under a Nikon microscope. The pathological diagnosis of normal, BCH, DYS and ESCC was performed on an adjacent H&E stained slide by two pathologists. In visual analysis, in situ hybridization staining intensity was graded as –, –/+, + and ++. Intense purple staining (+/++) was defined as positive. Background (–) or faint staining (–/+) was defined as negative. For digital image analysis, five images were picked randomly within encircled areas containing normal, BCH, DYS or ESCC at a magnification of x200. The mean optical densities from the positive signal and background were measured using Photoshop 5.0 program and the intensity ratio was calculated. We also measured the area with Imagine Plus 4.0 program to calculate the area ratio of the positively stained area to the total area. Finally, the expression index was calculated by multiplication of the intensity ratio and area ratio. The Student's t-test with unequal variance was used to compare the GRP expression index in different groups. Differences with P < 0.05 were regarded as significant.

Cell proliferation assay
ESCC cell lines, KYSE 150, 190, 450 and 510, were used to determine the effect of GRP on cell growth with the MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] assay. In brief, cells were seeded at 1 x 104 cells/well in a 96 multi-well plate with serum-free RPMI 1640 media. After 24 h culture in a 37°C, 5% CO2 incubator, the cells were treated with 1, 10, 100 and 1000 nM GRP for 48 h. Then the cells were incubated with 1 mg/ml MTT for 4 h at 37°C, the insoluble fomazan formed was dissolved in DMSO, and A550 nm was measured with an Emax Precision Microplate Reader (Molecular Devices, USA). Each group had six wells in replicate. The Student's t-test was used to analyze significant differences between the treatment and control groups.

Western blot
Cultured cells were collected, and protein was prepared with radioimmuno-precipitation assay lysis buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 0.4 mM phenylmethylsulfonyl fluoride, 0.3 µM aprotinin, 4 µM leupeptin, 3 µM pepstatin A and 10 µM indomethacin. The protein concentration was determined using Bradford analysis kit (Bio-Rad). Proteins (40 mg/lane) were loaded on 4–15% acrylamide gel and blotted onto a nitrocellulose membrane (Amersham, Heights, IL). After incubation in blocking buffer (5% non-fat milk), the membranes were incubated with mouse anti-human Cox-2 monoclonal antibody (1:1000) (Cayman, Ann Arbor, MI) overnight at 4°C. After washing with TBS containing 0.1% Tween 20, the membrane was then incubated with sheep anti-mouse horseradish peroxidase-labeled secondary antibody and visualized using the electrochemiluminescense detection kit (Amersham). The band intensities were determined with a densitometry (Bio-Rad). Average intensity was calculated from triple experiments.


    Results
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 Materials and methods
 Results
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 Supplementary material
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Differential display
Differential display was performed on the total RNA prepared from two pairs of esophageal tumors and adjacent non-tumorous epithelial samples. The displays of two of the 24 PCR reactions are shown in Figure 1. Some individual variability between the two non-tumorous epithelial samples or between the two tumor samples was also observed. All 17 differentially expressed bands between non-tumorous epithelial samples and tumors in 24 reactions were cut and re-amplified with the original primers. After sequencing and conducting a Blast search in the Gene Bank, 12 differentially expressed genes were identified (Table II). The differentially expressed genes were obtained consistently from two independent differential display experiments. Among the differentially expressed genes, seven sequences were genes with known names and functions, including GRP, phosphoglycerate mutase, P311 HUM (3.1), keratin 13, cystatin B, endoglin and annexin I; four were expressed sequence tags (ESTs), AA885875, AA807512, KIAA0648 and KIAA0112; and one had no EST available. Expressions of six genes, including GRP, phosphoglycerate mutase, P311 HUM (3.1), AA885875, AA807512 and the one with no EST available, were up-regulated. The other six genes, including keratin 13, cystatin B, endoglin, annexin I, KIAA0648 and KIAA0112, were down-regulated. Of these, GRP was selected for further studies.



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Fig. 1. Differentially expressed genes in human esophageal tumors. Total RNA was isolated from two human ESCC samples with adjacent non-tumorous esophageal epithelial tissues as the control. The primer combination in (A) is H-T11G anchor primer and AP4 arbitrary primer; in (B) is H-T11G and arbitrary primer AP6. Band 1, human gastrin-releasing peptide; band 2, unknown gene; band 3, human p311; band 4, unknown gene. N, total RNA prepared from adjacent non-tumorous esophageal epithelial tissues; T, total RNA from ESCC samples; 1, patient 1; 2, patient 2.

 

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Table II. Differentially expressed genes in ESCC in differential displaya

 
Over-expression of GRP in ESCC samples and cell lines
Expression of GRP was investigated using RT–PCR in 12 ESCC samples together with adjacent non-tumorous epithelial tissues for comparison (Figure 2). Ten (83.3%) cases had significant over-expression of GRP mRNA in tumor tissues over the paired non-tumorous samples (of these, only seven pairs of samples, #1, 3, 4, 6, 7, 8 and 9, are shown in Figure 2). Three out of the 12 (25.0%) adjacent non-tumorous samples expressed GRP mRNA; of which one had DYS (#5) and another two had BCH (only one sample, #2, is shown in Figure 2). In the cases shown, GRP expression in adjacent non-tumorous epithelium was more than the paired tumor. GRP expression was also detected in all four ESCC cell lines examined (data not shown). GRP mRNA expression levels of KYSE 150 and 450 cells were higher than KYSE 190 and 510 cells.



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Fig. 2. GRP over-expression in ESCC determined by RT–PCR. Total RNA was extracted from ESCC and adjacent non-tumorous esophageal epithelial tissues for RT–PCR. ß-Actin was used as an internal control. M, DNA marker; N, adjacent non-tumorous esophageal epithelial tissue; T, human ESCC.

 
GRP expression pattern in different stages of ESCC carcinogenesis
To determine the GRP expression pattern in different stages of esophageal squamous carcinogenesis, we analyzed GRP mRNA expressions using in situ hybridization in the frozen sections, including 16 normal esophageal epithelial samples, 21 BCH, seven DYS and 24 ESCC samples. Results showed that GRP was over-expressed in 17 out of 24 (70.9%) ESCC samples with moderate to strong positive staining (Table III). GRP was expressed in the cytoplasm and around the nuclei of positive cells distributed in the periphery or throughout the cancer nest in ESCC samples (Figure 3Ad). In nine out of 21 (42.8%) BCH and five out of seven (71.4%) DYS samples, GRP was expressed mostly in proliferating cells with moderate to strong staining intensity (Figure 3B) compared with the negative control (data not shown). Only three out of 16 (18.7%) normal epithelium weakly expressed GRP in the cytoplasm of cells that were located mainly in the basal and parabasal cell layers (Figure 3Ab). Mean expression index from digital image analysis revealed a gradual increase from normal to DYS (normal epithelium, 0.19; BCH, 1.23; DYS, 2.94). The mean expression index of ESCC (2.38) was similar to DYS (Table IV). All four ESCC cell lines, KYSE 150, 190, 450 and 510, also expressed strongly GRP mRNA. The representative positive staining of KYSE 150 cells is shown in Figure 3Af. Taken together, these results indicate that GRP was over-expressed in ESCC, and the expression progressively increased in BCH and DYS, suggesting that GRP is involved in the early stage of esophageal carcinogenesis.


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Table III. Visual scoring for GRP expression in esophageal samples with different lesions

 


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Fig. 3. GRP mRNA over-expression in ESCC and pre-cancerous lesions determined by in situ hybridization. DIG-labeled GRP cRNA probe was hybridized with human esophageal frozen sections and cultured monolayer ESCC cells. The sense probe was used as negative control. (A) a and b: normal esophageal epithelium (200x); c and d: ESCC (200x); e and f: ESCC cell line KYSE 150 (400x); a, c and e were hybridized with sense probe, and b, d and f with antisense probe. (B) g (BCH, 200x) and h (DYS, 200x) were hybridized with antisense probe. A color version of this figure is available as online supplementary material.

 

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Table IV. Results of digital image analysis for GRP expressiona

 
Stimulation effect of GRP on ESCC cell proliferation
To further study GRP functions in ESCC, we determined GRPR expression in ESCC cell lines, KYSE 150, 190, 450 and 510, using RT–PCR with full-length GRPR specific primers. Only KYSE 150 cells expressed GRPR mRNA, and the other three cell lines did not (Figure 4). When ESCC cell lines were treated with 10, 100 and 1000 nM GRP, the growth rate of KYSE 150 cells was increased in a dose-dependent manner with a 77% increase at 1000 nM (Figure 5). However, in KYSE 190, 450 and 510 cells, the addition of GRP did not affect cell growth. These results suggest that GRP stimulates ESCC cell proliferation in a GRPR-dependent manner.



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Fig. 4. GRPR expression in ESCC cell lines determined by RT–PCR. ß-Actin was used as an internal control. M, DNA marker; 1, 2, 3 and 4, human ESCC cell lines, KYSE 150, 190, 450 and 510, respectively.

 


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Fig. 5. GRP increased ESCC cell growth rate. Subconfluent, serum-starved human ESCC cell lines, KYSE 150, 190, 450 and 510 cells, were treated with 1, 10, 100 and 1000 nM GRP for 48 h, and then cell growth rate was determined with MTT assay. *Indicates significant difference (P < 0.01) between control and test groups. Values are presented as mean ± SD (n = 6).

 
Induction of Cox-2 protein expression by GRP on ESCC cells
In KYSE 150 cells, after treatment with 100 and 1000 nM GRP, Cox-2 protein expression levels increased. At 1000 nM GRP, the increase was 4.5-fold (Figure 6), and the mean increase was 3.4 ± 1.1-fold in triplicate experiments. In KYSE 510 cells, GRP treatment did not increase Cox-2 protein expression.



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Fig. 6. GRP induced Cox-2 protein expression in ESCC cells. Subconfluent, serum-starved human ESCC cell lines, KYSE 150 and 510 cells, were treated with 10, 100 and 1000 nM GRP for 6 h, and then protein expression levels were determined with western blot analysis. Representative pictures are shown. Band intensity was measured with densitometry and relative intensities (RI) are indicated.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
With differential display and sequencing, we found up- or down-regulated alterations of 12 genes in ESCC (Table II). The results on the over-expression of GRP and down-regulation of Keratin 13, Annexin I and Cystatin B are consistent with the results of our preliminary results with northern blot analysis (data not shown). Keratin 13 and Annexin I may be a differentiation marker in normally differentiated squamous epithelial cells (43,44), and their expressions are expected to be lower in ESCC, which are less differentiated. Cystatin B has been reported recently to be involved in esophageal tumor invasion and metastasis (45). GRP, a paracrine and autocrine growth factor involved in several tumors, appears to be the most interesting gene over-expressed in ESCC. Therefore, GRP was selected for further study. The other genes may be subjects for future investigation.

GRP over-expression in ESCC was confirmed by RT–PCR. Over-expression of GRP was observed in 10 out of 12 ESCC samples and in a few adjacent non-tumorous samples with pre-cancerous lesions. High expression levels of GRP were observed in all four ESCC cell lines studied. Our results from in situ hybridization studies showed that GRP mRNA existed in the cytoplasm, and the positive cells were distributed in the periphery or throughout the cancer nest in ESCC. ESCC is known to be developed through a multistage process including BCH and DYS; severe DYS is considered an immediate precursor of ESCC (17). The observation that GRP was over-expressed progressively from BCH to DYS and ESCC (Table IV), suggests that GRP is involved in the early stage of esophageal squamous carcinogenesis. This is the first demonstration of GRP over-expression in esophageal carcinogenesis.

In the present study, most normal epithelium did not express GRP, but some normal epithelial samples expressed GRP in cells located in the basal cell layer (Figure 3Ab). The cells in the basal layer of the esophageal epithelium have proliferation potential and they are frequently hyperproliferative in many individuals in the high-risk population in Linzhou, China. Therefore, GRP-positive cells in the basal cell layer in apparently normal epithelium may represent cells that start to proliferate. GRP may function as an autocrine growth factor for esophageal epithelial cells similar to that demonstrated for fetal lung development (20). Staniek et al. demonstrated the presence of GRP and GRPR on basal and parabasal layer cells of human skin, suggesting that GRP modulates epidermal cell functions (46). Recently, GRP was reported to promote re-epithelialization during cutaneous wound healing (47).

The present results showed that GRP expression was closely associated with cell hyperproliferation, and increased in BCH, DYS and ESCC during esophageal squamous carcinogenesis. GRP significantly increased the cell proliferation rate of the GRPR-positive KYSE 150 cells. The most well-studied GRP-induced signal transduction pathway involves AP-1 activation, which promotes cell proliferation (35). Activated AP-1 gene may also elevate Cox-2 expression (36). The induction of Cox-2 expression by GRP was demonstrated in GRPR-positive ESCC cells (Figure 6), suggesting GRP can activate the Cox-2 pathway, which can contribute to cell proliferation and tumorigenesis (48).

In summary, the present results show that GRP is over-expressed in ESCC and suggest that GRP is involved in early stage esophageal squamous carcinogenesis. GRP stimulates cell growth and increases Cox-2 protein expression in GRPR-positive ESCC cells. More detailed research is needed to further elucidate the role of GRP function in esophageal carcinogenesis and enhancing arachidonic acid metabolism. Considering GRP is a small peptide, one of the possibilities may be to assess the GRP levels in serum samples from patients or high-risk population for biomarker evaluation. Our cell line study also revealed the important role of GRPR in cell proliferation. Monoclonal antibodies against GRP and GRPR antagonists have been utilized in a clinical trial in lung cancer (3739). Our results may lead to practical applications in the development of preventive or therapeutic agents for human ESCC.


    Supplementary material
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 Abstract
 Introduction
 Materials and methods
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 Supplementary material
 References
 
Supplementary material can be found at: http://www.carcin.oupjournals.org/


    Acknowledgments
 
We would like to thank Dr Xiaoxin Chen for his helpful discussions. Supported by NIH Grant CA65781 and facilities from NIEHS Center Grant ES 05022 and NCI Cancer Center Supporting Grant CA 72030.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

  1. Macfarlane,G.J. and Boyle,P. (1994) The epidemiology of esophageal cancer in the UK and other European countries. J. Roy. Soc. Med., 87, 334–337.[ISI][Medline]
  2. Yang,C.S. (1980) Research on esophageal cancer in China: a review. Cancer Res., 40, 2633–2644.[Abstract]
  3. Shi,S.T., Yang,G.Y., Wang,L.D., Xue,Z., Feng,B., Ding,W., Xing,E.P. and Yang,C.S. (1999) Role of p53 gene mutations in human esophageal carcinogenesis: results from immunohistochemical and mutation analyses of carcinomas and nearby non-cancerous lesions. Carcinogenesis, 20, 591–597.[Abstract/Free Full Text]
  4. Xing,E.P., Yang,G.Y., Wang,L.D., Shi,S.T. and Yang,C.S. (1999) Loss of heterozygosity of the Rb gene correlates with pRb protein expression and associates with p53 alteration in human esophageal cancer. Clin. Cancer Res., 5, 1231–1240.[Abstract/Free Full Text]
  5. Shi,S.T., Feng,B., Yang,G.Y., Wang,L.D. and Yang,C.S. (1996) Immunohistoselective sequencing (IHSS) of p53 tumor suppressor gene in human esophageal precancerous lesions. Carcinogenesis, 17, 2131–2136.[Abstract]
  6. Yang,G., Zhang,Z., Liao,J., Seril,D., Wang,L., Goldstein,S. and Yang,C.S. (1997) Immunohistochemical studies on Waf1p21, p16, pRb and p53 in human esophageal carcinomas and neighboring epithelia from a high-risk area in northern China. Int. J. Cancer, 72, 746–751.[CrossRef][ISI][Medline]
  7. Xing,E.P., Nie,Y., Song,Y., Yang,G.Y., Cai,Y.C., Wang,L.D. and Yang,C.S. (1999) Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma. Clin. Cancer Res., 5, 2704–2713.[Abstract/Free Full Text]
  8. Xing,E.P., Nie,Y., Wang,L.D., Yang,G.Y. and Yang,C.S. (1999) Aberrant methylation of p16INK4a and deletion of p15INK4b are frequent events in human esophageal cancer in Linxian, China. Carcinogenesis, 20, 77–84.[Abstract/Free Full Text]
  9. Nie,Y., Yang,G., Song,Y., Zhao,X., So,C., Liao,J., Wang,L.D. and Yang,C.S. (2001) DNA hypermethylation is a mechanism for loss of expression of the HLA class I genes in human esophageal squamous cell carcinomas. Carcinogenesis, 22, 1615–1623.[Abstract/Free Full Text]
  10. Hollstein,M.C., Smits,A.M., Galiana,C., Yamasaki,H., Bos,J.L., Mandard,A., Partensky,C. and Montesano,R. (1988) Amplification of epidermal growth factor receptor gene but no evidence of ras mutations in primary human esophageal cancers. Cancer Res., 48, 5119–5123.[Abstract]
  11. Victor,T., Du Toit,R., Jordaan,A.M., Bester,A.J. and van Helden,P.D. (1990) No evidence for point mutations in codons 12, 13, and 61 of the ras gene in a high-incidence area for esophageal and gastric cancers. Cancer Res., 50, 4911–4914.[Abstract]
  12. Hollstein,M.C., Peri,L., Mandard,A.M., Welsh,J.A., Montesano,R., Metcalf,R.A., Bak,M. and Harris,C.C. (1991) Genetic analysis of human esophageal tumors from two high incidence geographic areas: frequent p53 base substitutions and absence of ras mutations. Cancer Res., 51, 4102–4106.[Abstract]
  13. Casson,A.G., Wilson,S.M., McCart,J.A., O'Malley,F.P., Ozcelik,H., Tsao,M.S. and Chambers,A.F. (1997) ras mutation and expression of the ras-regulated genes osteopontin and cathepsin L in human esophageal cancer. Int. J. Cancer, 72, 739–745.[CrossRef][ISI][Medline]
  14. Miyazaki,S., Sasno,H., Shiga,K., Sawai,T., Nishihira,T., Okamoto,H. and Mori,S. (1992) Analysis of c-myc oncogene in human esophageal carcinoma: immunohistochemistry, in situ hybridization and northern and Southern blot studies. Anticancer Res., 12, 1747–1755.[ISI][Medline]
  15. Kan,T., Shimada,Y., Sato,F., Maeda,M., Kawabe,A., Kaganoi,J., Itami,A., Yamasaki,S. and Imamura,M. (2001) Gene expression profiling in human esophageal cancers using cDNA microarray. Biochem. Biophys. Res. Commun., 286, 792–801.[CrossRef][ISI][Medline]
  16. Hu,Y.C., Lam,K.Y., Law,S., Wong,J. and Srivastava,G. (2001) Identification of differentially expressed genes in esophageal squamous cell carcinoma (ESCC) by cDNA expression array: overexpression of Fra-1, Neogenin, Id-1, and CDC25B genes in ESCC. Clin. Cancer Res., 7, 2213–2221.[Abstract/Free Full Text]
  17. Lam,A.K. (2000) Molecular biology of esophageal squamous cell carcinoma. Crit. Rev. Oncol. Hematol., 33, 71–90.[ISI][Medline]
  18. Spindel,E.R., Chin,W.W., Price,J., Rees,L.H., Besser,G.M. and Habener,J.F. (1984) Cloning and characterization of cDNAs encoding human gastrin-releasing peptide. Proc. Natl Acad. Sci. USA, 81, 5699–5703.[Abstract]
  19. Naylor,S.L., Sakaguchi,A.Y., Spindel,E. and Chin,W.W. (1987) Human gastrin-releasing peptide gene is located on chromosome 18. Somat. Cell. Mol. Genet., 13, 87–91.[ISI][Medline]
  20. Bunnett,N. (1994) Gastrin-releasing peptide. In Walsh,J.H. and Dockray,G.J. (eds) Gut Peptides: Biochemistry, and Physiology. Raven Press, New York, pp. 423–445.
  21. Kroog,G.S., Jensen,R.T. and Battey,J.F. (1995) Mammalian bombesin receptors. Med. Res. Rev., 15, 389–417.[ISI][Medline]
  22. Price,J., Penman,E., Wass,J.A. and Rees,L.H. (1984) Bombesin-like immunoreactivity in human gastrointestinal tract. Regul. Pept., 9, 1–10.[CrossRef][ISI][Medline]
  23. Battey,J., Wada,E., Corjay,M. et al. (1992) Molecular genetic analysis of two distinct receptors for mammalian bombesin-like peptides. J. Natl Cancer Inst. Monogr., 13, 141–144.[Medline]
  24. Jensen,J.A., Carroll,R.E. and Benya,R.V. (2001) The case for gastrin-releasing peptide acting as a morphogen when it and its receptor are aberrantly expressed in cancer. Peptides, 22, 689–699.[CrossRef][ISI][Medline]
  25. Ferris,H.A., Carroll,R.E., Lorimer,D.L. and Benya,R.V. (1997) Location and characterization of the human GRP receptor expressed by gastrointestinal epithelial cells. Peptides, 18, 663–672.[CrossRef][ISI][Medline]
  26. Charitopoulos,K.N., Lazaris,A.C., Aroni,K., Kavantzas,N., Nikolakopoulou,E. and Davaris,P. (2000) Immunodetection of gastrin-releasing peptide in malignant melanoma cells. Melanoma Res., 10, 395–400.[CrossRef][ISI][Medline]
  27. Radulovic,S.S., Milovanovic,S.R., Cai,R.Z. and Schally,A.V. (1992) The binding of bombesin and somatostatin and their analogs to human colon cancers. Proc. Soc. Exp. Biol. Med., 200, 394–401.[Abstract]
  28. Sun,B., Halmos,G., Schally,A.V., Wang,X. and Martinez,M. (2000) Presence of receptors for bombesin/gastrin-releasing peptide and mRNA for three receptor subtypes in human prostate cancers. Prostate, 42, 295–303.[CrossRef][ISI][Medline]
  29. Sun,B., Schally,A.V. and Halmos,G. (2000) The presence of receptors for bombesin/GRP and mRNA for three receptor subtypes in human ovarian epithelial cancers. Regul. Pept., 90, 77–84.[CrossRef][ISI][Medline]
  30. Siegfried,J.M., Krishnamachary,N., Gaither Davis,A., Gubish,C., Hunt,J.D. and Shriver,S.P. (1999) Evidence for autocrine actions of neuromedin B and gastrin-releasing peptide in non-small cell lung cancer. Pulm. Pharmacol. Ther., 12, 291–302.[CrossRef][ISI][Medline]
  31. Carney,D.N., Cuttitta,F., Moody,T.W. and Minna,J.D. (1987) Selective stimulation of small cell lung cancer clonal growth by bombesin and gastrin-releasing peptide. Cancer Res., 47, 821–825.[Abstract]
  32. Cuttitta,F., Carney,D.N., Mulshine,J., Moody,T.W., Fedorko,J., Fischler,A. and Minna,J.D. (1985) Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. Nature, 316, 823–826.[ISI][Medline]
  33. Wakelam,M.J., Davies,S.A., Houslay,M.D., McKay,I., Marshall,C.J. and Hall,A. (1986) Normal p21N-ras couples bombesin and other growth factor receptors to inositol phosphate production. Nature, 323, 173–176.[ISI][Medline]
  34. Millar,J.B. and Rozengurt,E. (1990) Arachidonic acid release by bombesin. A novel postreceptor target for heterologous mitogenic desensitization. J. Biol. Chem., 265, 19973–19979.[Abstract/Free Full Text]
  35. Kim,H.J., Evers,B.M., Litvak,D.A., Hellmich,M.R. and Townsend,C.M.,Jr (2000) Signaling mechanisms regulating bombesin-mediated AP-1 gene induction in the human gastric cancer SIIA. Am. J. Physiol. Cell Physiol., 279, C326–334.[Abstract/Free Full Text]
  36. Guo,Y.S., Hellmich,M.R., Wen,X.D. and Townsend,C.M.,Jr (2001) Activator protein-1 transcription factor mediates bombesin-stimulated cyclooxygenase-2 expression in intestinal epithelial cells. J. Biol. Chem., 276, 22941–22947.[Abstract/Free Full Text]
  37. Koppan,M., Halmos,G., Arencibia,J.M., Lamharzi,N. and Schally,A.V. (1998) Bombesin/gastrin-releasing peptide antagonists RC-3095 and RC-3940-II inhibit tumor growth and decrease the levels and mRNA expression of epidermal growth factor receptors in H-69 small cell lung carcinoma. Cancer, 83, 1335–1343.[CrossRef][ISI][Medline]
  38. Jungwirth,A., Pinski,J., Galvan,G., Halmos,G., Szepeshazi,K., Cai,R.Z., Groot,K., Vadillo-Buenfil,M. and Schally,A.V. (1997) Inhibition of growth of androgen-independent DU-145 prostate cancer in vivo by luteinising hormone-releasing hormone antagonist Cetrorelix and bombesin antagonists RC-3940-II and RC-3950-II. Eur. J. Cancer, 33, 1141–1148.[CrossRef][Medline]
  39. Kelley,M.J., Linnoila,R.I., Avis,I.L., Georgiadis,M.S., Cuttitta,F., Mulshine,J.L. and Johnson,B.E. (1997) Antitumor activity of a monoclonal antibody directed against gastrin-releasing peptide in patients with small cell lung cancer. Chest, 112, 256–261.[Abstract/Free Full Text]
  40. Tanaka,H., Shimada,Y., Imamura,M., Shibagaki,I. and Ishizaki,K. (1997) Multiple types of aberrations in the p16 (INK4a) and the p15(INK4b) genes in 30 esophageal squamous-cell-carcinoma cell lines. Int. J. Cancer, 70, 437–442.[CrossRef][ISI][Medline]
  41. Fang,M.Z., Mar,W.C. and Cho,M.H. (2001) Cadmium-induced alterations of connexin expression in the promotion stage of in vitro two-stage transformation. Toxicology, 161, 117–127.[CrossRef][ISI][Medline]
  42. Xu,X.C. (2001) Detection of altered retinoic acid receptor expression in tissue sections using in situ hybridization. Histol. Histopathol., 16, 205–212.[ISI][Medline]
  43. Jetten,A.M., George,M.A., Smits,H.L. and Vollberg,T.M. (1989) Keratin 13 expression is linked to squamous differentiation in rabbit tracheal epithelial cells and down-regulated by retinoic acid. Exp. Cell Res., 182, 622–634.[ISI][Medline]
  44. Paweletz,C.P., Ornstein,D.K., Roth,M.J. et al. (2000) Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma. Cancer Res., 60, 6293–6297.[Abstract/Free Full Text]
  45. Wang,L.D., Lipkin,M., Qui,S.L., Yang,G.R., Yang,C.S. and Newmark,H.L. (1990) Labeling index and labeling distribution of cells in esophageal epithelium of individuals at increased risk for esophageal cancer in Huixian, China. Cancer Res., 50, 2651–2653.[Abstract]
  46. Staniek,V., Misery,L., Peguet-Navarro,J., Sabido,O., Cuber,J.C., Dezutter-Dambuyant,C., Claudy,A. and Schmitt,D. (1996) Expression of gastrin-releasing peptide receptor in human skin. Acta. Derm. Venereol., 76, 282–286.[ISI][Medline]
  47. Yamaguchi,Y., Hosokawa,K., Nakatani,Y., Sano,S., Yoshikawa,K. and Itami,S. (2002) Gastrin-releasing peptide, a bombesin-like neuropeptide, promotes cutaneous wound healing. Dermatol. Surg., 28, 314–319.[CrossRef][ISI][Medline]
  48. Tripathi,S.C., Vosseller,K.A. and McCormick,D.L. (1996) Arachidonic acid metabolism and cell proliferation in rat mammary carcinoma cells treated with indomethacin. Biochim. Biophys. Acta, 1316, 5–7[ISI][Medline]
Received May 27, 2003; revised December 12, 2003; accepted January 13, 2004.





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