**4. Acetic acid-induced gastric ulcer healing and** *COX* **mRNA levels in Mongolian gerbils with or without** *H. pylori* **infection**

*H. pylori* infection in humans is best modeled in Mongolian gerbils (MGs), and chronic infection with *H. pylori* induces inflammatory cell infiltration in the gastric mucosa in MGs (**Figure 3A**). It was previously shown that *H. pylori* infection significantly delayed acetic acid-induced ulcer healing in mice and MGs [27–29]. *H. pylori* infection induces aberrant DNA methylation of several CGIs in MGs [30]. As described above, COX-2-derived PGs are important for gastric ulcer healing, and the *COX-2* promoter is densely methylated in the human gastric mucosa in the presence of *H. pylori* infection. To investigate the roles of *COX-2*

**Figure 3.** A. Microscopic features of *H. pylori*-infected gastric mucosa of Mongolian gerbils (MGs). Sections were stained with hematoxylin and eosin. B. Gross appearance 10 days after gastric ulcer induction of *H. pylori*-infected MGs. C. Microphotograph 10 days after gastric ulcer induction in *H. pylori*-infected MGs. D. Serial changes in acetic acidinduced gastric ulcer areas in MGs with or without *H. pylori* infection. *H. pylori* (ATCC43504; American Type Culture Collection, Rockville, MD) was grown in Brucella broth (Becton Dickinson, Cockeysville, MD) containing 10% v/v horse serum for 40 h at 37°C under microaerobic conditions (15% CO2 ) and high humidity with shaking (150 rpm). Male MGs (MGs/Sea) were purchased from Kyudo (Saga, Japan). At 11 weeks of age, *H. pylori* (0.8 mL samples of Brucella broth containing 1.0 × 109 colony-forming units) was delivered intragastrically using an oral catheter after fasting for 24 h. Gastric ulcers were induced experimentally in MGs according to the method described by Wang et al. [40]. Briefly, after anesthetization with ketalar, the abdomen was opened through a midline incision, and 50 μL of 25% acetic acid was injected in the subserosa of the anterior wall of the stomach. The MGs were killed at5, 10, and 20 days after ulcer induction, and the stomach was dissected and removed. The maximum and minimum diameters of the ulcers were measured, and the ulcer area, which was approximately elliptical, was calculated and was compared between MGs with and without *H. pylori* infection. Values are the mean ± SE. The animal care committee of St. Marianna University approved the experimental design, and the animals were cared for in accordance with institutional guidelines.

**Figure 2.** Effects of a PKC stimulator (α-phorbol 12,13-dibutyrate; PDBu) on COX-2 mRNA expression with or without 5-aza-dC in the human gastric adenocarcinoma cell line KATO-III. KATO-III cells were treated with vehicle (1 μmol/L)

**Figure 1.** *COX-2* DNA methylation levels in patients with or without *H. pylori* infection, and patients previously with

with or without 5-Aza-dC for 5 days. The figure is modified from Ref. [26].

successfully eradicated *H. pylori* infection. The figure is modified from Ref. [26].

210 Chromatin and Epigenetics

methylation and *H. pylori* infection in gastric ulcer healing, *COX* mRNA levels in samples prepared from acetic acid-induced gastric ulcers in MGs were examined. Then, the effects of *COX-2* methylation on *COX-2* mRNA expression were also investigated *in vitro* using an *H. pylori-*infected MG stomach-derived cell line.

**5. Effects of** *COX-2* **methylation on its mRNA expression in MGs** 

As discussed above, *COX-2* mRNA expression is regulated by an epigenetic mechanism in KATO-III human gastric carcinoma cells in which *COX-2* is densely methylated [22]. To investigate the role of methylation in *COX-2* mRNA expression in *H. pylori*-infected gastric mucosa of MGs, we treated MGC2 cells with 5-aza-dC, a methyltransferase inhibitor, or trichostatin A (TSA), a histone deacetylase inhibitor. MGC2 is an adenocarcinoma cell line established from the gastric cancer tissue of a *H. pylori*-infected MG [32]. *COX-2* mRNA expression levels in these cells were restored after the addition of 5-aza-dC. In contrast, treatment with TSA did not induce *COX-2* mRNA expression (**Figure 5**). These results indicated that *COX-2* mRNA expression in MGs is regulated via both transcriptional and epigenetic mechanisms. Histone acetylation was not involved in silencing *COX-2* expression

Role of *COX-2* Promoter Methylation and *Helicobacter pylori* Infection in Impaired Gastric Ulcer…

http://dx.doi.org/10.5772/intechopen.79973

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*in vitro*

in these cells.

5% CO2

**6. Cloning the MG** *COX-2* **promoter region**

or without 5-Aza-dC (1 μmol/L) or TSA for 5 days. Values shown are the mean ± SE.

As mentioned above, human *COX-2* has abundant CGIs in the promoter region. To examine whether CGIs are present in the *COX-2* promoter region of MGs, we performed genomic

**Figure 5.** Effects of 5-aza-dC, a methyltransferase inhibitor, or trichostatin A (TSA), a histone deacetylase inhibitor, on *COX-2* mRNA expression in MG gastric adenocarcinoma MGC2 cells. The MGC2 cells [32] were a generous gift from Dr. Tatematsu and were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) plus serum expander MITO (0.1%, Collaborative Biomedical Products Bedford, MA, USA) on a type I collagen-coated dish (Asahi Techno Glass, Japan). All the cultures were incubated at 37°C with 95% air and

. *COX-2* mRNA expression was measured by real-time PCR in the cell lines grown in the presence of vehicle with

Gastric ulcers were produced by injecting 25% of acetic acid (0.03 mL) into the submucosal layer of the gastric wall of the antral-oxyntic border in MGs 48 weeks after inoculation with an *H. pylori* suspension in Brucella broth. The MGs were killed at 5, 10, and 20 days after ulcer induction, and the stomachs were dissected and removed. The maximum and minimum diameters of the ulcers were measured, and the area of each ulcer, which was approximately elliptical, was calculated and compared between MGs with and without *H. pylori* infection. The ulcer area was largest on day 5, and then gradually decreased. In accordance with previous reports [29], the gastric ulcer area was larger in *H. pylori*-infected MGs than in uninfected MGs (**Figure 3B**–**D**). While *COX-2* mRNA expression at the ulcer edge was increased 5 days after acetic acid injection in MGs without *H. pylori* infection, as was reported in rats and mice [31], no increases in *COX-2* mRNA levels were observed in *H. pylori*-infected MGs. In contrast, gastric ulceration was not associated with a change in *COX-1* mRNA levels in MGs with or without *H. pylori* infection (**Figure 4A,B**). Thus, *H. pylori* infection caused delayed ulcer healing and impaired COX-2 induction in MG stomachs.

**Figure 4.** *COX* mRNA levels during healing of an acetic acid-induced gastric ulcer in MGs with or without *H. pylori* infection. *COX* mRNA levels in acetic-acid-induced gastric ulcers in MGs were measured by real-time PCR. First-strand cDNA was prepared by reverse transcription of 5 μg of total RNA using superscript III reverse transcriptase (Applied Biosystems, Carlsbad, CA, USA). Real-time quantitative reverse transcription PCR was carried out using TaqMan Gene Expression Assays with a 7500 real-time PCR system (Applied Biosystems) according to the manufacturer's instructions. Primers for *COX-1*, *COX-2*, and *β-actin* were designed based on their cDNA sequences (GenBank accession nos. AB 044783, AB044784, and AB040445, respectively) and according to previous reports [33]. SDS2.1 software (Applied Biosystems) was used to perform the comparative Δ-Ct analysis. Glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control. Values are the mean ± SE. β-Actin left: gctacagcttcaccaccaca, right: ccatctcttgctcgaagtcc, 93 bp. COX-1 left: gtggctatttcctgcagctc, right: agtgggtgccagtggtagag, 112 bp. COX-2 left: tgggcgtgaaaggaaataag, right: ggggatcagggatgaacttt, 87 bp.
