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Keywords:

  • gastrin;
  • p16;
  • anion exchanger 1;
  • gastric antrum cancer

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Our previous studies demonstrated that expression and interaction of p16 with anion exchanger 1 (AE1) in gastric cancer cells is correlated with progression and shorter survival of the cancer. In this article, the effects of gastrin on p16 and AE1 and its implication in prevention and treatment of gastric cancer were studied by molecular biology techniques, animal experiment and clinical analysis. The results showed that expression of p16 in human gastric body carcinoma was downregulated along with the progression of the cancer, suggesting the reverse correlations between gastrin and p16 in vivo. Further experiments indicated that gastrin suppressed the expression of p16 via the p16 promoter and thereafter resulted in the degradation of AE1 in gastric cancer cells. Silencing of AE1 or p16 significantly inhibited the proliferation of the cancer cells. Using a xenograft tumor model in nude mice, we showed that experimental systemic hypergastrinemia induced by the administration of omeprazole led to decreased expression of AE1 and p16 as well as to a marked growth inhibition of SGC7901 tumors. It is concluded that a moderate plasma gastrin level is beneficial to the growth inhibition of gastric cancer by suppressing the expression of AE1 and p16. This finding may have an important implication for the prevention and treatment of cancers arise in the gastric antrum.

Anion exchanger 1 (AE1) is abundantly expressed in erythrocytes and efficiently contributes to intracellular pH regulation and homeostasis of erythrocytes by catalyzing the reversible exchange of Cl/HCOmath image across the plasma membrane.1, 2 Structural analysis has revealed that AE1 consists of a N-terminal cytoplasmic domain, a multispanning membrane domain and an acidic short C-terminal cytoplasmic domain.3 The C-terminal region of AE1 contains the binding sites for tumor suppressor p16, which has been reported in our previous paper.4

The p16 negatively regulates the cell cycle. Abnormal expression of p16 has been demonstrated in a variety of cancer cells.5–8 Functional inactivation of the p16 gene induced by several factors has been shown in many different types of human carcinomas.9–11 However, recent studies revealed that overexpression of the p16 protein in cytoplasm of cancer cells is associated with the tumor progression and poor prognosis, suggesting that cytoplasmic distribution of p16 might indicate an inactive form of the protein.12–15 Our previous studies demonstrated that expression and interaction of p16 with AE1 in gastric cancer cells is correlated with progression and shorter survival of the cancer.12, 16 Thus, how AE1/p16 becomes involved in the carcinogenesis of gastric cancer needs further elucidation.

Gastrin is expressed primarily in G cells located in the gastric antrum and to a lesser in the duodenum and is involved in gastric acid production.17 Another physiologic role of gastrin is in regulating the proliferation of gastric mucosal cells which has led to investigations into its effects on stimulating tumor cell growth.18 It has been reported that gastrin plays a role in gastric cancer development.19 Overexpression of human gastrin in specific transgenic animals leads to the development of precancerous conditions identified by histopathology, immunohistochemistry and biomolecular technology. Thus, gastrin is considered to be the accelerant for cell proliferation and migration mediated by the gastrin receptor.20 However, recent studies have demonstrated that gastrin suppresses the growth of gastrin receptor-expressing colon cancer cells.21, 22 In addition, overexpression of gastrin in transgenic mice results in apoptosis of parietal cells, accompanied by increased expression of Bax and downregulation of Bcl-2.23 In clinical investigations, both increased and decreased levels of plasma gastrin have been observed in gastric cancer.24–26 The contradictory results suggest that variable effects of gastrin on cancer with different types develop at different stages. In this study, we determine the correlations between gastrin and AE1 and p16, and explore their potential roles and implications for the prevention and treatment of cancers arise in the gastric antrum.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Tissue specimens and immunohistochemical staining

Tumor specimens were fixed in 10% formalin and embedded in paraffin. For detection of p16 expression in primary gastric body carcinoma, 86 gastric body cancer samples (25 samples in early and 61 samples in advanced stage) were collected from surgical resection and endoscopic biopsy. To observe the expression of gastrin in gastric antrum carcinoma and para-carcinoma, 44 gastric antrum cancer samples (22 samples in early and 22 samples in advanced stage) were also obtained from surgical resection and endoscopic biopsy. In addition, xenograft tumor samples were subjected to immunohistochemistry to detect the expression of AE1 and p16. Sections were cut to a thickness of 4 μm and deparaffinized by immersion in xylene for 40 min. Endogenous peroxidase was blocked by immersion in 3% hydrogen peroxidase in methanol for 10 min. After overnight incubation at 4°C with the primary rabbit anti-human gastrin polyclonal antibody (MAIXIN-Bio, Fuzhou, China), rabbit anti-human p16 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-human AE1 monoclonal antibody (Sigma, St. Louis, MO), the slices were washed in PBS and then incubated for 15 min with second antibody. DAB was performed to develop color and Mayer's hematoxylin was used for counterstaining. Normal mouse IgG2a and rabbit IgG isotypes were applied as negative control instead of primary anti-AE1, gastrin and p16 antibodies, respectively.

Cell culture and western blot

The following cell lines were used in the study: gastric cancer cell line SGC7901, human embryonic kidney cell line HEK293 and breast cancer cell line MCF-7. All cell lines used in the experiments were purchased from the Cell Bank of Shanghai Institute of Cell Biology of the Chinese Academy of Sciences. Cells were maintained at 37°C in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 throughout. Cell lysate samples were separated on a SDS-PAGE (8–12%) by electrophoresis and transferred onto nitrocellulose membranes. All blots were blocked 1 hr with 5% nonfat dry-powdered milk (W/V) in TBS-T buffer and incubated with anti-p16 (Santa Cruz Biotechnology) or anti-AE1 antibodies (Sigma, St. Louis, MO or a gift from Dr. M.L. Jennings, University of Arkansas, Fayetteville, AR, USA). Blots were incubated with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 hr at room temperature. The immunoreactive bands were visualized by enhanced chemiluminescence reagent (Pierce, Rockford IL). To study the effects of gastrin (G17, Sigma, St. Louis, MO) on gastric cancer cells, SGC7901 cells were cultivated in vitro and treated with gastrin in different concentrations.

RT-PCR and real-time PCR

Total RNA was collected from SGC7901 cells. RNA samples were reverse-transcribed into cDNA using random hexamer primers and ReverTra Ace-α-reverse transcriptase (Toyobo, Osaka, Japan). For measuring p16 or AE1, 400 ng of cDNA in a total volume of 50 μl for each reaction was amplified by a KOD PCR kit (Toyobo, Osaka, Japan). The primers used for real-time PCR were as follows: p16 forward (5′-CCCCCACTACCGTAAATGTCCAT-3′), p16 reverse (5′-CTGCCATTTGCTAGCAGTGTGACT-3′), AE1 forward (5′-ACCACATCACACCCGGGTA-3′), AE1 reverse (5′-ACCAACGTGGCCTCTGAATC -3′), GAPDH forward (5′-CCAGAACATCATCCCTGCCT-3′) and GAPDH reverse (5′-CCTGCTTCACCACCTTCTTG-3′). Quantitative PCR was performed using the Bio-Rad iCycler Realtime PCR System using 10 μmol/l each primer and SYBR Green I detection reagent (Takara).

Plasmid construction

The p16 promoter (808 bp fragment, −876/−68 bp relative to the initiator codon ATG) was cloned into the luciferase reporter gene vector, pGL3-basic (Promega, Madison WI). Three 5′-truncated sequences (−623/−68 bp, −426/−68 bp and −223/−68 bp) of the p16 promoter were generated by PCR and cloned into the luciferase reporter gene vector, pGL3-basic. The fidelities of the constructs were confirmed by sequencing.

To generate the p16 siRNA vectors, three-target sequences were selected as follows: I, 5′-CGCACCGAATAGTTACGGT-3′; II, 5′-ACCAGAGGCAGTAACCATG-3′; and III, 5′-AGAACCAGAGAGGCTCTGA-3′. To generate the AE1 siRNA vectors, three-target sequences of AE1 siRNA were selected as follows: I, 5′-GGGTACTGTTCTCCTAGAC-3′; II, 5′-CGGTCTAGAGTACATCGTG-3′; and III, 5′-GGCAACCTTTGATGAGGAG-3′. These sequences were synthesized and inserted into the pSIREN-RetroQ neo vector (Cloneteck, Palo Alto, CA) in accordance with the manufacturer's instructions.

Luciferase assay

SGC7901 cells were transiently transfected with the p16-luc vector. To normalize for transfection efficiency, the cells were cotransfected with a pRL-TK reporter construct. Eight hours post-transfection, the Opti-MEM was replaced with DMEM medium containing 10% FBS in the presence or absence of gastrin in different concentrations. The Dual-luciferase report assay was performed after 48 hr in accordance with the manufacturer's instructions.

MTT assay and cell doubling time analysis

3-(4,5-Dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to detect the cell viability. SGC7901 cells were seeded into 96 well plates and incubated at 37°C for 24 hr. The cells were then incubated with gastrin or not. After a further time in culture, 10 μl of MTT (5 mg/ml, Sigma) was added to each well and incubated again for 4 hr at 37°C. MTT was then removed and 150 μl of dimethylsulphoxide was added to each well. The absorbance values were measured at a wavelength of 490 nm using an ELISA reader. To analyze cell doubling time, SGC7901 cells were plated in 24 well plates and treated with gastrin in different concentrations for 7 days. After then, cells were reseeded at a starting density of 3 × 104 cells per well in DMEM medium supplemented with 10% FBS. The cells were treated for another 6 days. The number of cells at various time points was evaluated using a hemocytometer. Mean cell doubling time (MCDT) was calculated as given below: MCDT = t[log 2/(logNt − logN0)], where N0 is the cell number at time zero, Nt is the cell number at time final and t is time difference between N0 and Nt, respectively.

Cell cycle analysis

SGC7901 cells were seeded at a density of 3 × 104 in 60 mm culture dishes. Subsequently, the cells were treated with 10−7 or 10−10 mol/l of gastrin for 12 days in complete medium. After treatment with gastrin, cells were harvested by trypsinization, centrifuged at 2000 rpm for 5 min, washed in PBS and resuspended in cold 75% ethanol. Cells were labeled with propidium iodide (0.05 mg/ml) in the presence of RNase A (0.5 mg/ml) and incubated at room temperature in the dark for 30 min. DNA contents were analyzed using a flow cytometer.

Generation of AE1 and p16 knockdown cell lines

We next used RNA interference to generate stable knockdown of p16 or AE1 in SGC7901 cells, respectively. SGC7901 cells were transfected with pSIREN-RetroQ-AE1II, pSIREN-RetroQ-p16I, or pSIREN-RetroQ-control retrovirus. One milliliter of culture medium containing the integrated retrovirus from the host HEK293 cells was added 1 hr after SGC7901 plating. The same procedure was repeated after 24 hr. Seventy-two hours later, 1 μg/ml of puromycin (Sigma) was added to the plates for screening. After 1 week of selection, the surviving cells were isolated and maintained on selection medium until used.

Tumor model and therapy

The tumor growth response of SGC7901 cells to gastrin was assessed in a xenograft nude mouse model. Female athymic BALB/c nude mice (4–6 weeks old) were purchased from Shanghai Experimental Animal Centre, Chinese Academy of Science and the experiments were approved by the animal research committee in Shanghai Jiao Tong University. Systemic hypergastrinemia was induced by treatment of omeprazole, a proton pump inhibitor that increases serum gastrin levels 2- to 4-fold, as described in other experiments.21, 27 The nude mice were subcutaneously injected with 4 × 106 SGC7901 cells suspended in 0.2 ml 0.9% NaCl into flank. Once palpable tumors were established and reached ∼300 mm3, animals were randomized into two groups (n = 8 per group). One group served as control received administration of 0.9% NaCl daily. The other group was treated with omeprazole (75 mg/kg, i.p). The tumor volume (V) was calculated based on the formula: V = length (mm) × width2 (mm)/2. All mice were killed after administration for 12 days.

To investigate the effects of AE1-targeted siRNA on gastric cancer growth, in another experiment, 12 xenograft nude mice were prepared. Seven days after inoculation, the mice were divided randomly into 3 groups. Mice were then treated with (s.c. around tumors) injections of siRNA (2.5 μmol/l, Shanghai Genepharma Co.)/0.5% atelocollagen complex (final atelocollagen concentration, 0.25%), nonspecific control (2.5 μmol/l)/0.5% atelocollagen complex and natural saline/0.5% atelocollagen every 3 days for a total of 7 injections. Tumor size was measured in 2 dimensions using a caliper, and tumor volume (mm3) was calculated as a2 × b/2 mm3 (a, minor axis; b, major axis).

Statistical analysis

All the data are given as the mean ± S.D. SPSS 13.0 statistical package (SPSS, Chicago, IL) was used to analyze the experiment data. Multiple comparisons were done with one-way analysis of variance (ANOVA). Two group comparisons were performed with a t-test. Comparisons of proportions were evaluated using a chi-square test. Statistical differences with p values less than 0.05 were considered to be significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Gradual downregulation of p16 in primary gastric body carcinoma

It is generally accepted that damage to the gastric mucosa leads to gastritis and mucosa atrophy, which is considered to be an essential step in gastric tumorigenesis. Atrophic gastritis can be divided into A and B types. Type A gastritis involves mainly the gastric body and fundus due to the presence of serum antiparietal anti-intrinsic factor antibodies. Clinical studies showed that the hypergastrinemia was common in the patients with type A gastritis and gastric body cancer.28, 29 To clarify the relationship between gastrin and p16 in vivo, we first detected the expression of p16 in primary gastric body carcinoma by immunohistochemistry (86 cases, 25 early and 61 advanced gastric cancer patients). The results showed that the p16 protein was abundantly expressed in gastric cancer cell and mainly located in cytoplasm, compared with that in surrounding normal tissue (Figs. 1a1c). The p16 expression with a frequency of 80% (20 of 25 cases) in the early stage and declined to 52.5% (32 of 61 cases) in the advanced stage of the gastric cancer (Fig. 1d). The results suggest that there is an inverse relationship between gastrin level and p16 expression.

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Figure 1. Expression frequency of p16 in primary gastric body carcinoma developed at different stages. Expression of p16 was detected by immunohistochemistry and more than 10% expression was considered to be positive, regardless of the staining intensity. (a) Negative or feeble staining of p16 in para-cancer gastric tissue (×100). (b) Strong positive staining of p16 in gastric carcinoma (×100). (c) High magnification of the arrow-marked area in (b) (×400). The cytoplasmic and nuclear expression of p16 was observed. (d) Statistic analysis of p16 expression frequency at different progressing stages of gastric body carcinoma. n represents the positive specimen number.

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Gastrin suppresses the expression of p16 in gastric cancer cells

To determine the effects of gastrin on p16 expression, the gastric cancer SGC7901 cells were treated with different concentrations of gastrin. As shown in Figure 2, gastrin dose-dependently downregulated the p16 expression (Fig. 2a). Gastrin, at 10−7 mol/l, significantly decreased the expressions of p16 mRNA and protein in SGC7901 cells from 6 hr post-treatment (Figs. 2b and 2c). Quantitative analysis by real-time PCR showed that the mRNA level of p16 was decreased to 60% at 24 hr after gastrin treatment (Fig. 2d). To determine whether gastrin affected the stability of the p16 protein, the pEGFP-p16 construct was transfected into HEK293 cells. The expression of GFP-p16 protein was not changed by 10−7 mol/l gastrin, indicating that gastrin reduced the expression of p16 by decreasing transcription of p16, but not by influencing p16 protein stability (Fig. 2e).

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Figure 2. Gastrin dose- and time-dependently suppressed the expression of endogenous p16 in gastric cancer SGC7901 cells. (a) SGC7901 cells were treated with indicated dose of gastrin. Endogenous p16 protein was detected by Western blot. (b) SGC7901 cells were treated with 10−7 mol/l gastrin for different time as indicated. Endogenous p16 protein was detected by Western blot. (c) and (d) Detection and quantitative analysis of p16 mRNA in SGC7901 cells by RT-PCR or real-time-PCR, respectively. SGC7901 cells were treated with 10−7 mol/l gastrin for different time as indicated. (e) HEK293 cells were treated with or without 10−7 mol/l gastrin after transfection of pEGFP-p16 constructs. Exogenous p16 expression was analyzed by Western blot at the indicated time.

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Gastrin decreases p16 promoter activity

As shown in Figure 3a, promoter of p16 gene contains 5 putative GC boxes, named GC-I to V. The p16 promoter was highly active in SGC7901 cells compared with cells transfected with the pGL3-basic empty vector alone. Gastrin (10−7 mol/l, 48 hr) exhibited about 44% inhibition in luciferase activity in cells transiently expressing p16-luc (−876/−68 bp) compared with untreated controls. To further determine which region of p16 promoter is responsible for gastrin-mediated inhibition of p16 promoter activity, a series of 5′-truncated p16-reporter constructs were generated in pGL3-basic vector containing progressive deletion of the 5′-end of the full length p16 promoter construct, p−876/−68.30 The deletion construct, p−623/−68, showed similar inhibition (58%), indicating that deletion from −876 bp to −623 bp did not alter the inhibitory effects of gastrin on p16 promoter activity. However, further deletion to p−426/−68 and p−223/−68 attenuated the inhibitory effects of gastrin. These results suggested that the gastrin-responsive element is located between the −623 and −426 region (Fig. 3b). Furthermore, we examined the effect of different concentrations of gastrin on p16 promoter activity and the results showed that gastrin in lower concentrations (10−12 to 10−9 mol/l) suppresses the p16 transcription in a dose-dependent manner, but showed an invariable decrease of p16 transcription in response to higher concentrations of gastrin (10−8 to 10−7 mol/l) (Fig. 3c).

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Figure 3. Gastrin suppressed p16 promoter activity. (a) Schematic diagram of the 876 bp human p16 promoter. This region contains 5 GC boxes and a negative regulatory element associated with a 24 kDa protein. The numbers indicate the position relative to the ATG (+1). (b) SGC7901 cells were transfected with luciferase reporter vectors containing different truncations of the p16 promoter: −876 bp, −623 bp, −426 bp or −223 bp relatively to the ATG (left). Corresponding relative luciferase activities treated with gastrin (solid bar) or without gastrin (control, hollow bar) were determined by reporter gene assay (right). Bars show fold increase in luciferase activity for the p16-reporter constructs cloned into pGL3 vector compared with promoter-less pGL3-basic vector (negative control). Data are representative of experiments performed 3 times in triplicate. *p < 0.01, compared with control. (c) SGC7901 cells were transfected with p16 luciferase reporter vectors (p16-876 constructs) and treated with gastrin in different concentrations. Relative promoter activities to control were determined by reporter gene assay at 48 hr post treatment. *p < 0.05, compared with control.

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Gastrin-induced degradation of AE1 is dependent on the downregulation of p16

Our previous studies showed that the p16 protein directly bound to the AE1 C-terminus and both proteins were expressed in gastric cancer cells.4, 12, 16 Therefore, we further investigated the effects of gastrin on AE1 expression. As shown in Figure 4, gastrin significantly decreased the expression of AE1 protein but not mRNA in SGC7901 cells. Together, the results indicated that gastrin reduced the expression of AE1 and p16 via different mechanisms. We presume that gastrin decreased the expression of p16 protein and the absence of p16 resulted in the degradation of AE1. To test this hypothesis, the pcDNA3.0-AE1 and pEGFP-p16 constructs were cotransfected into MCF-7 cells, a breast cancer cell line expressing neither AE1 nor p16 protein.31 Western blotting showed that the expression of AE1 protein was significantly increased, while AE1 and p16 were coexpressed, and compared with the control experiment, the cells were cotransfected with pcDNA3.0-AE1 and p16 empty vector (p < 0.01, Figs. 5a and 5b). To confirm the influence of p16 on AE1 stability, the MCF-7 cells were cotransfected with pcDNA3.0-AE1 and pEGFP-p16 or p16 empty vector and the cells were incubated in cycloheximide (25 μg/ml), which blocks de novo protein synthesis. Cellular extracts were prepared at different times after the addition of cycloheximide and the expression of AE1 protein was detected by Western blot. As shown in Figures 5c and 5d, in the presence of cycloheximide, overexpression of p16 resulted in a slow rate of AE1 degradation compared with cells transfected with p16 empty vector, suggesting that expression of p16 protein increases the stability of AE1 protein via direct interaction (Supporting Information File 1). The half-life of the AE1 protein in the cells was extended from 12 to 24 hr as a consequence of p16 overexpression.

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Figure 4. Gastrin-induced degradation of AE1 in SGC7901 cells. SGC7901 cells were treated with 10−7 mol/l gastrin for the indicated time. (a) AE1 protein expression was determined by Western blot. (b) AE1 mRNA transcription was determined by RT-PCR.

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Figure 5. Expression of p16 increased the stability of AE1 protein. (a) MCF-7 cells were cotransfected with pcDNA3.0-AE1 and pEGFP-p16 or pEGFP-c1 constructs. Cells were harvested 48 hr post transfection and the exogenous AE1 and p16 were analyzed by Western blot. (b) The relative abundance of AE1 protein was analyzed by using the Quantity one 4.4.0 software (n = 3). (c) MCF-7 cells were cotransfected with pcDNA3.0-AE1 and pEGFP-p16 or pEGFP-c1 constructs. Cycloheximide (CHX) was added 24 hr post transfection and the expression of AE1 protein at the indicated time was analyzed by Western blot. (d) Expression amount of AE1 was evaluated by using the same software described above (n = 3). The values were obtained by comparing the density of AE1 band at different time points to that at zero point. The error bars represent mean ± SD.

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Effects of gastrin on proliferation of SGC7901 cells

To investigate the role of AE1 and p16 in proliferation of gastric cancer cells and to evaluate their role in gastrin-induced cell proliferation, SGC7901 cells were treated as follows: (1) cells were treated with 10−7 or 10−10 mol/l gastrin, respectively; (2) cells were stably transfected with siRNAII for AE1, siRNAI for p16 and scramble fragments. As shown in Figures 6a and 6b, siRNA fragments efficiently blocked the expression of AE1 or p16 in SGC7901 cells, respectively. Stable silencing of AE1 or p16 significantly inhibited the viability of SGC7901 cells compared with scramble transfections (Fig. 6c), indicating that the expression of cytoplasmic AE1 and p16 favoring growth of gastric cancer cells. Silencing of AE1 resulted in the return of p16 to the nucleus and played a role in negatively regulating the cell cycle, thus cell viability was inhibited. On the other hand, silencing of p16 also inhibited the viability of SGC7901 cells, suggesting that the cytoplasmic p16 does not play a role in negative regulation of the cell cycle in gastric cancer cells (Supporting Information File 2), but rather interrupting the regulation pathway of the cell cycle. In contrast, overexpression of AE1 in SGC7901 cells promoted the cell proliferation (Fig. 6d). The results are consistent with our clinical analysis, in which the expression of AE1 was correlated with deeper invasion and shorter survival of the cancer patients,32 suggesting that the gastrin inhibit cell viability through downregulating AE1/p16 pathway. Long term treatment with gastrin (10−7 mol/l or 10−10 mol/l) induced significant inhibition of SGC7901 cell viability. Gastrin (10−10 mol/l) also efficiently downregulated the expression of p16 in SGC7901 cells (Figs. 6e and 6f). The doubling time of SGC7901 cells was increased from 36.2 (control) to 40.5 hr (10−10 mol/l gastrin) and 41.8 hr (10−7 mol/l gastrin) (Fig. 7a). Cell cycle analysis demonstrated that at 12 days after gastrin treatment (10−7 or 10−10 mol/l), the S and G2 populations were remarkably increased, whereas the G1 population was decreased (Fig. 7b), suggesting that gastrin caused cell cycle arrest at S-phase.33

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Figure 6. Viability of SGC7901 cells in different conditions. (a) and (b) SGC7901 cells were stably transfected with siRNA constructs as indicated and the expression of endogenous p16 or AE1 was detected by western blot. (c) The expression of p16 and AE1 in SGC7901 cells was stably blocked by siRNA I or siRNA II, respectively. The relative cell viability was determined by MTT assay. *p < 0.05, compared with NC-transfected cells. (d) SGC7901 cells were transfected with pcDNA3.0-AE1 or empty vectors. The cells were selected by G418 for 1 week and then seeded. The relative cell survival ratio to the control was determined by MTT. *p < 0.05, compared with control or cells transfected with empty vector. (e) and (f) Cells were treated with 10−7 or 10−10 mol/l gastrin for different days and the relative cell survival ratio was determined by MTT assay. The values are expressed as mean ± SD of 3 determinations. *p < 0.05, compared with control. Western blot showed that 10−10 mol/l gastrin efficiently downregulated the expression of p16 in SGC7901 cells.

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Figure 7. Growth inhibition and S-phase arrest of SGC7901 cells induced by gastrin. (a) SGC7901 cells treated with gastrin for 7 days (10−7 mol/l or 10−10 mol/l) were reseeded at a density of 3 × 104 in 24 well plates and exposed to 10−7 mol/l or 10−10 mol/l gastrin for 6 days. The cells were cultured and harvested at different time points after trypsinization. Cell suspensions were placed onto a hemocytometer and cell numbers were counted using the trypan blue dye exclusion assay. All the data are presented as means ± SD and are the results of three individual experiments. (b) SGC7901 cells were treated with 10−7 or 10−10 mol/l gastrin for 12 days. Cell cycles were analyzed by flow cytometry. Points mean value of 3 separate experiments. *p < 0.05, compared with control.

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Systemic hypergastrinemia and AE1-targeted siRNA in a nude mice tumor model leads to growth inhibition in SGC7901 tumors

To verify the effects of gastrin on p16 and AE1, and on the growth inhibition in vivo, nude mice were subcutaneously inoculated with gastric SGC7901 cells, and systemic hypergastrinemia was induced by omeprazole treatment which was reported in recent papers.21, 34 As shown in Figures 8a and 8b, systemic hypergastrinemia induced by the administration of omeprazole led to decreased expression of AE1 and p16 as well as to a marked growth inhibition of SGC7901 tumors. These results indicated that the omeprazole-induced gastrin secretion leads to a decrease of p16 and AE1 expression which is benefit to growth inhibition of gastric cancer cells. Also, the AE1-targeted siRNA significantly inhibited the growth of the tumor (Fig. 8c).

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Figure 8. Systemic hypergastrinemia or AE1-targeted siRNA treatment in a nude mice tumor model leads to growth inhibition in SGC7901 tumors. Nude mice were inoculated with gastric SGC7901 cells and after 2 weeks, animals were randomized and treated with either vehicle (0.9% NaCl) or omeprazole to induce systemic hypergastrinemia. (a) Tumor sizes were measured and calculated. Relative growth rates were calculated for each growing tumor to allow comparison of rates among different-sized tumors. The percentage increase in tumor volume was defined as the relative tumor volume on days 3, 5, 7, 9 and 11 normalized to the initial tumor volume on the day of treatment (day 0). *p < 0.01, compared with 0.9% NaCl treatment group. (b) Tumors were excised and submitted to immunohistochemistry analysis with p16 antibody or AE1 antibody respectively. (a′) and (c′) The p16 and AE1 staining in paraffin tissue section of 0.9% NaCl-treated tumors. (b′) and (d′) The p16 and AE1 staining in paraffin tissue section of omeprazole-treated tumors (×100). (c) To achieve RNAi-based tumor therapy, a AE1-targeted siRNA was directly injected into SGC7901 tumors. The tumors in nude mice were prepared as described earlier. Seven days after inoculation, the mice were divided randomly into 3 groups and treated with siRNA, nonspecific control (NC) and normal saline (NS), respectively. Tumor size was measured at the indicated time points. *p < 0.05, compared with NC or NS group; **p < 0.01, compared with NC or NS group.

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To further explore potential clinical applications of the experimental data, we detected the expression of gastrin in gastric antrum carcinoma by immunohistochemistry (44 cases, 22 early and 22 advanced gastric antrum cancer samples obtained from surgical resection). The expression of gastrin was negative in all carcinoma tissues and it was detected in the para-carcinoma tissues with different frequencies. In the early stage of gastric antrum cancer, positive staining of gastrin was detected in 16 (72.7%) of 22 cases, whereas it was detected in 3 (13.6%) of 22 cases in the advanced stage of the cancer (Fig. 9), suggesting that the expression of gastrin in para-carcinoma tissues was gradually decreased along with the progression of gastric antrum cancer.

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Figure 9. Expressions of gastrin in gastric carcinoma and para-carcinoma. (a) Positive staining of gastrin in G cells around cancer tissue in gastric antrum. (b) Negative staining of gastrin in gastric antrum carcinoma (×100). (c) Expression frequency of gastrin at different progressing stages of gastric antral carcinoma. n represents the negative or positive specimen number.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

To date, a number of downstream target genes of gastrin have been reported, however, no document has described the correlation of p16 with gastrin, even though p16 is a key factor in cell cycle regulation. In this article, we show that gastrin regulates the expression of p16 through the promoter of the p16 gene in a dose- and time-dependent manner and the sequence of the gastrin-responsive element is located between the −623/−68 bp and −426/−68 bp regions. The results are consistent with the previous report indicating that a negative regulatory element located at −491 to −485 bp in the p16 promoter and a 24-kDa protein is related to the negative regulation.30 Decreased expression of AE1 protein, but not mRNA, was coinstantaneously observed, indicating that the interaction of p16 with AE1 increased the stability of AE1 protein and gastrin-induced downregulation of AE1 is not due to the inhibition of transcription, but rapid degradation of AE1 protein. The data demonstrated that the expression of AE1 and p16 are under the regulation of gastrin and the three factors affecting gastric carcinogenesis and cancer progression.

It is well known that the inactivation of the p16 results in excessive cell proliferation.35–37 However, in the stomach, p16 is expressed at a very low level and that is beneficial for repair of gastric epithelial damage. This study shows that p16 is upregulated in the cytoplasm, but not nucleus of gastric cancer cells and the expression of p16 had regional differences in gastric carcinoma. In the cancer arise in gastric body region, the frequency of p16 expression declined significantly from the early stage to the advanced stage of gastric cancer, whereas it was maintained, or even increased in gastric antrum cancer along with cancer progression (data not shown). This is correlated with the concentration of gastrin in the plasma and intercellular spaces. Namely, in gastric antrum cancer, the expression of gastrin was decreased and the plasma gastrin was reduced because there were significant correlation between the integrated plasma gastrin secretion and the antral gastrin content in patients with gastric cancer. In contrast, in gastric body or fundus cancer, gastrin was continuously secreted to the plasma due to the low-acidity stimulus and the p16 expression was inhibited.

Control of cell proliferation is important for cancer prevention since cell proliferation has an essential role in carcinogenesis. The p16 negatively regulates the cell cycle and is commonly considered to act in the nucleus.38 The present data show that silencing of p16 inhibits the proliferation of gastric cancer cells, suggesting that the p16 in gastric cancer cells does not normally regulate the cell cycle, but rather interrupts the cell cycle pathway. This is consistent with our previous study, in which both cytoplasmic AE1 and p16 are correlated with the progression of gastric cancer and the p16 protein was transferred from the nucleus to the cytoplasm along with development of gastric cancer.16, 32

Gastric cancer can be divided into 2 histologic types, the intestinal and the diffuse type. The intestinal type is somewhat more common than the diffuse type and about 60 to 80% of intestinal-type cancers arise from the gastric antrum. Studies have shown that a reduced plasma gastrin is found in 30% of patients with gastric cancer and most of these abnormalities are confined to patients with the intestinal type.39 Our present results indicate that gastrin disappeared in gastric antrum carcinoma, but was retained in para-cancer tissues (72.7%, 16 of 22 cases, in the early stages and 13.6%, 3 of 22 cases, in advanced stages), suggesting that the expression of gastrin is decreased along with gastric cancer progression. It is reasonable to conclude that there are synchronous low concentrations of gastrin in the plasma and intercellular spaces in patients with gastric antrum cancer. The results suggest that application of gastrin may have a beneficial effect for antigastric cancer strategies via suppression of p16 and AE1. Systemic hypergastrinemia and AE1-targeted siRNA treatment in nude mice suppressed the growth of SGC7901 tumors, indicated that a moderate plasma gastrin level is beneficial to the growth inhibition of gastric cancer by suppressing the expression of AE1 and p16. This finding may have an important implication for the prevention and treatment of cancers arise in the gastric antrum.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Professor M. L. Jennings ML (University of Arkansas, Arkansas, USA) for providing the anti-AE1 antibody.

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  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_25124_sm_suppfig1.tif2213KSupporting Information Figure 1.
IJC_25124_sm_suppfig2.tif2638KSupporting Information Figure 2.

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