Eradication of Helicobacter pylori decreases the expression of p53 and c-Myc oncogenes

Introduction 

Helicobacter pylori (H. pylori) may cause many gastroduodenal diseases including acute gastritis, atrophic gastritis, intestinal metaplasia, peptic ulcer and others disorders. Although many studies revealed close association between gastric cancer and H. pylori, there have been only few studies that report on gastric carcinogenesis associated with chronic H. pylori [1], [2].

H. pylori is a class 1 gastric carcinogen. However, it remains unclear whether H. pylori affects molecular alterations in chronic gastritis. It is well known that only a minority of H. pylori-positive patients with chronic gastritis develop gastric cancer, and so the link is unclear [2].

Nardone et al. [3] had stated that gastric carcinogenesis is a multi-step process progressing from chronic gastritis through glandular atrophy, metaplasia, and dysplasia. Acquired genomic instability, generally precedes neoplastic clonal expansion. H. pylori damages stimulates gastric cell proliferation, which leads to mucosal repair, but which can also induce cellular DNA damage. The most frequent epiphenomenon of DNA alteration is activation of oncogenes and/or mutation of oncosuppressor genes. The role of these genes has been studied in gastric carcinogenesis, but their interrelation with H. pylori infection has yet to be defined. Thus, this study was designed to detect the relation between H. pylori and gastric carcinogenesis and to investigate the effect of its eradication on the expression of p53 and c-Myc in H. pylori-associated chronic gastritis.

As a transcription factor, p53 has roles in regulation of proliferation, apoptosis, genomic repair, controls the onset of cellular senescence and suppresses angiogenesis [4], [5]. p53 mutation is among the major episodes in the multi-step process of gastric carcinogenesis, while it has also been reported in pre-malignant lesions of the stomach, such as chronic atrophic gastritis, intestinal metaplasia, and dysplasia [1].

c-Myc is a basic transcriptional factor that regulates several genes involved in cell proliferation, differentiation, apoptosis, and oncogenesis. c-Myc expression was studied in gastric cancer with H. pylori infection by many authors [6], [7]. Also, the relationship between H. pylori infection and c-Myc expressions was investigated in a series of diseases and it was concluded that in gastric carcinogenesis, H. pylori might cause the imbalance between proliferation and apoptosis in precancerous lesions, leading to tumour-suppressor system mutation and telomerase reactivation, and finally causes gastric cancer [8].

Patients and methods 

A total of 55 chronic gastritis patients (38 male and 17 female with mean age: 57.1years) underwent endoscopic examination for evaluation of dyspypsia. H. pylori positive were 45 cases while H. pylori negative were only 10 cases. All H. pylori-positive patients had successful eradication therapy (15 days of treatment with omeprazole 20mg twice a day, clarithromycin 500mg twice a day, and tinidazole 500mg twice a day), and underwent biopsies before and six months after eradication.

H. pylori in the stomach was detected by rapid urease test and histological examination. For the urease test, biopsy specimens were immediately inserted into the rapid urease test solution. H. pylori was histologically detected by May-Giemsa stain. H. pylori eradication was considered successful when the results of the two tests (both the urase test and Giemsa stain) were found negative.

Biopsy specimens were taken from five points of the stomach, as recommended by the updated Sydney system [9], i.e. the lesser curvature of the antrum, the greater curvature of the antrum, the smaller curvature of the angle, the lesser curvature of the middle corpus and the greater curvature of the upper corpus. All biopsies were fixed in buffered formalin for 24h and embedded in paraffin. Serial sections were stained with haematoxylin-eosin and May-Giemsa stain. The status of the gastric mucosa was evaluated according to the updated Sydney system. The degree of inflammation (manifested by mild lymphocyte and plasma-cell infiltration), neutrophil activity, atrophy (glandular morphology absent in the mucosa and replaced by connective tissue, inter-glandular space was infiltrated by plasma cells and lymphocytes), dysplasia (nuclear atypia with or without architectural abnormalities in the gastric epithelium, but without invasion) and intestinal metaplasia (presence of goblet cells in gastric mucosa) were classified into four grades, with 0 for ‘normal’, 1 for ‘mild’, 2 for ‘moderate’, and 3 for ‘marked’ changes.

The following procedure was performed for Immunohistochemical detection of p53 and c-Myc. Serial paraffin sections were washed in 1/15mol/L phosphate buffered saline (pH 7.4) three times for five minutes, and pre-incubated in normal rabbit serum (1:10 in PBS) for 20min. Next, sections were incubated with primary antibodies for 16h at 4°C, followed by the avidin–biotin complex method. The sections were immersed in 0.05mol/L Tris–HCl buffer containing 0.02% 3,3′-diaminobenzidine tetrahydrochloride and 0.005% H2O2, and the nuclei were counterstained with haematoxylin. Control sections incubated with normal mouse IgG instead of the primary antibody showed no non-specific staining. The primary antibodies used in this study for p53, staining the sections were incubated with anti-rabbit or mouse antibody (Envision HRP, Dako Company) for 10min and anti-c-Myc protein (Oncogene Science, San Diego, California, USA; c-Myc p62, dilution 1:50). After antigen retrieval (for c-Myc in 1mM EDTA (pH 8.0), sections were incubated overnight at 41C with monoclonal mouse antibodies to c-Myc (1:100 dilution, Abcam Inc., Cambridge, MA, USA). Immunoreactivity was detected using the DAB Map Kit (PIERCE, Woburn, MA, USA), based on the avidin–biotin complex immunoperoxidase technique.

The degree of immunopositivity was evaluated semiquantitatively. A total of 300 cells were counted in random fields from representative areas of the lesions and the immunoreactive cells were roughly assessed and expressed as percentages. The scoring system for both antibodies tested was: 0–5% (negative: −); 5–25% (low positivity: +); 25–50% (moderate positivity: ++); >50% (high positivity: +++) according to Nardone et al. [3].

SPSS 10.0 was used for statistical analysis. Statistical analysis was performed with t-test or χ2-test, with statistical significance indicated by a value of p<0.05.

Results 

The study included 55 cases, 45 cases of these were H. pylori positive. The neutrophilic infiltrate was detected only in H. pylori positive cases and the majority of cases were grade 3, 26 cases were positive for lymphoplasmacytic infiltrate; 20 of them were H. pylori positive, 23 cases showing atrophy; 20 of them were H. pylori positive and the remaining three cases were H. pylori negative. These were of grade 1. Intestinal metaplasia were seen in 16 cases; 15 of them were H. pylori positive (Fig. 1). Glandular dysplasia was detected only in 10 cases; all of them were H. pylori positive. The degree of activity of chronic gastritis is summarized in Table 1.

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Fig. 1. Severe intestinal metaplasia in H. pylori positive case as the metaplastic epithelium is present in the middle (H&E stain, 200×).

Table 1. Activity of chronic gastritis in the studied cases.

		PNLs n=38				Lymphocytes n=26				Atrophy n=23				IM n=16				Dysplasia n=10				Sig.
		0	1	2	3	0	1	2	3	0	1	2	3	0	1	2	3	0	1	2	3
	H. pylori (−) N=10	10 (100%)	0	0	0	4 (40%)	1 (10%)	2 (20%)	3 (30%)	7 (70%)	3 (30%)	0	0	9 (90%)	1 (10%)	0	0	10 (100%)	0	0	0	0.03
	H. pylori (+) N=45	7 (15%)	10 (22%)	12 (27%)	16 (36%)	25 (56%)	10 (22%)	7 (15%)	3 (7%)	25 (56%)	11 (24%)	8 (18%)	1 (2%)	30 (67%)	7 (15%)	6 (13%)	2 (5%)	35 (77%)	3 (7%)	4 (9%)	3 (7%)	0.04
	Sig.	0.001				0.03				0.04				0.01				0.02

Table 2 summarizes the correlation between the histological variables and the studied oncogens. Concerning p53, the positive cases were 12 and all of them were H. pylori positive, 4 of them were +, 5 were ++ and 3 were +++ (Fig. 2). There was a significant difference between the staining positivity of p53 and all the studied variables including neutrophilic infiltrate, lymphoplasmacytic infiltrate, gastric glandular atrophy, intestinal metaplasia and dysplasia. According to c-Myc staining, the total positive cases were 10 and all of them were H. pylori (+) cases, 4 were +, 3 were ++ as seen in (Fig. 3) and 3 were +++. Again there was a significant correlation between the staining positivity and the grades of the studied variables. As the grade of chronic gastritis activity increases, the expression positivity of both p53 and c-Myc increases with significant difference between the grades (Table 2).

Table 2. Correlation between p53 and c-Myc and the studied histological variables in H. pylori (+) cases.

			PNLs				Lymphocytes				Atrophy				IM				Dysplasia				Total
			0	1	2	3	0	1	2	3	0	1	2	3	0	1	2	3	0	1	2	3
	p53 (n=12)	−	7	8	7	11	25	6	2	0	25	6	2	0	30	3	0	0	31	2	0	0	33
		+	0	2	2	0	0	3	1	0	0	2	2	0	0	1	3	0	4	0	0	0	4
		++	0	0	3	2	0	1	3	1	0	3	2	0	0	3	1	1	0	1	3	1	5
		+++	0	0	0	3	0	0	1	2	0	0	2	1	0	0	2	1	0	0	1	2	3
	Sig.		0.003				0.02				0.01				0.02				0.004				55
	c-Myc (n=10)	−	7	8	8	12	25	7	3	0	25	5	5	0	30	3	2	0	31	2	0	0	35
		+	0	2	2	0	0	2	2	0	0	4	0	0	0	2	2	0	2	0	2	0	4
		++	0	0	2	1	0	1	2	0	0	1	2	0	0	1	2	0	0	1	1	1	3
		+++	0	0	0	3	0	0	0	3	0	1	1	1	0	0	1	2	0	0	1	2	3
	Sig.		0.01				0.009				0.03				0.005				0.03				55

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Fig. 2. H. pylori positive case showing +++ p53 expression. This case showed moderate intestinal metaplasia. Note the intense inflammatory infiltrate. (strept avidin–biotin–DAB, 400×).

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Fig. 3. c-Myc moderate expression in a case of H. pylori positive case. The positivity is mainly in the intestinal metaplasia portion. (strept avidin–biotin–DAB, 200×).

After treatment and complete resolution of H. pylori infection in, the same histopathological variables and immunohistochemical staining of the studied oncogenes were evaluated. The activity of chronic gastritis was measured and summarized in Table 3. We found less case positive for neutrophilic infiltrate, lymphocytic infiltrtate, atrophy, intestinal metaplsia (Fig. 4) and even glandular dysplasia after treatment. The grading of all these parameters became less (Table 3) with significant difference between before and after treatment in same cases.

Table 3. Activity of chronic gastritis in studied cases after treatment.

		PNLs n=18				Lymphocytes n=8				Atrophy n=9				Intestinal metaplasia n=7				Dysplasia n=7
		0	1	2	3	0	1	2	3	0	1	2	3	0	1	2	3	0	1	2	3
	Treated cases N=45	27 (60%)	15 (33%)	3 (7%)	0	37 (82%)	4 (9%)	3 (7%)	1 (2%)	36 (80%)	5 (11%)	4 (9%)	0	38 (84%)	3 (7%)	3 (7%)	1 (2%)	38 (84%)	3 (7%)	2 (4%)	2 (4%)
	Sig.	0.004				0.003				0.005				0.009				0.007

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Fig. 4. A case of inteastinal metaplasia grade 3 persisting after treatment of H. pylori (H&E stain, 400×).

Regarding p53 results after treatment, the number of positive cases became only seven instead of 12, three cases were +, three cases were ++ (Fig. 5) and only one case was +++. There was a positive correlation between the p53 and all the variables of chronic gastritis as summarized in Table 4.

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Fig. 5. p53 Expression after treatment in a case of chronic gastritis. Expression was graded as +++ and became ++ after treatment (strept avidin–biotin–DAB, 400×).

Table 4. Correlation between p53 and c-Myc and the studied histological variables after treatment.

			PNLs				Lymphocytes				Atrophy				Intestinal metaplasia				Dysplasia				Total
			0	1	2	3	0	1	2	3	0	1	2	3	0	1	2	3	0	1	2	3
	P53 (n=7)	−	27	11	0	0	37	1	0	0	36	2	0	0	38	0	0	0	38	0	0	0	38
		+	0	3	0	0	0	2	1	0	0	2	1	0	0	2	2	0	0	2	1	0	3
		++	0	1	2	0	0	1	2	0	0	1	2	0	0	1	1	0	0	1	1	1	3
		+++	0	0	1	0	0	0	0	1	0	0	1	0	0	0	0	1	0	0	0	1	1
	Sig.	0.01					0.01				0.03				0.02				0.01				45
	c-Myc(n=6)	−	27	11	1	0	37	1	1	0	36	3	0	0	37	1	1	0	38	1	0	0	39
		+	0	2	1	0	0	3	0	0	0	2	1	0	1	1	1	0	0	2	1	0	3
		++	0	1	1	0	0	0	2	0	0	0	2	0	0	1	1	0	0	0	1	1	2
		+++	0	1	0	0	0	0	0	1	0	0	1	0	0	0	0	1	0	0	0	1	1
	Sig.	0.03					0.04				0.04				0.02				0.01				45

Concerning c-Myc, the number of positive cases were found to be only 6 after treatment compared to 10 before treatment; three cases were + (Fig. 6), 2 cases were ++ and only one case was +++. A positive correlation between c-Myc and all the variables of chronic gastritis was found and is summarized in Table 4.

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Fig. 6. c-Myc expression after treatment. Expression was reduced from ++ to become + only (strept avidin–biotin–DAB, 400×).

Discussion 

c-Myc and p53 expression are the most widely used markers of genomic instability [10]. In this work, c-Myc and p53 expression were not detected in H. pylori negative cases as previously reported by van der Hulst et al. [11].

We have found the grade of positive immunostaining increases with the progress of lesions from active chronic gastritis to atrophic gastritis to intestinal metaplasia to dysplasia. Increased p53 expression may be an important molecular event involved in the early stage of gastric carcinogenesis. This justifies its use as biomarker to assess risk for the development of gastric carcinoma. Similar reports came from other authors [1], [4], [12] who have reported that H. pylori positive gastritis especially that accompanied with intestinal metaplasia showed a higher p53 expression. Anagnostopoulos et al. [13], however, reported that p53 was expressed only in cases with high grade dysplasia and not in pre-dysplastic stages and concluded that p53 mutation is a late event during the development of gastric cancer. Our results confirm the relationship between atrophic and/or metaplastic gastric mucosa with H. pylori infection and p53 expression.

Marinone et al. [14] data indicate that irreversible genetic changes in the p53 protein does not occur in non-neoplastic gastric mucosa with metaplasia and H. pylori related chronic gastritis and they concluded that the increase in p53 levels is due to an increased production of the wild-type protein probably related to an inflammatory response induced by H. pylori infection.

In this work, H. pylori negative cases were completely negative for c-Myc expression while H. pylori infection was associated with positive expression of c-Myc. This is in contrast to reports by other authors [15] who found no evidence of expression of c-Myc in any gastritis sample. c-Myc has been reported to be increased in H. pylori-associated gastritis and is associated with increased cell proliferation.

In other studies [6], [7] it was reported that in H. pylori-associated gastritis, there is downregulation of p27 and increased c-Myc, the net result of which is increased cell proliferation. Furthermore, H. pylori may influence both telomerase activity and c-Myc expression in chronic atrophic gastritis.

In Zhan et al. [16] study, expression of c-Myc was significantly higher in carcinoma than that in dysplasia than in metaplasia. Also the expression of c-Myc in metaplastic cases and dysplasia with H. pylori infection was significantly higher than in those without infection; in this work, c-Myc was completely negative in cases without H. pylori infection.

After eradication treatment, we found a decrease in the number of cases with neutrophilic and lymphocytic infiltration and even in the same case there was a decrease in the grade of the inflammatory infiltrate. Also the number and the grade of atrophy, intestinal metaplasia and dysplasia were significantly decreased.

The effects of H. pylori eradication on atrophy and intestinal metaplasia are controversial in previous studies. Some authors [17], [18] have reported results similar to the present work. The patchy nature of the lesions and the subjective nature of the interpretation may account for the controversial findings. Our finding of disappearance or regression of atrophy after H. pylori eradication suggests that inflammatory infiltration plays a role in this controversial histological finding. On the other hand, some authors [11] document no changes in intestinal metaplasia and atrophy after H. pylori eradication, while others [19] reported that H. pylori eradication does not reduce the histologic metaplasia score, but changes the cellular phenotype of metaplasia. This change of phenotype may be an important factor in the reduction of cancer incidence after eradication of H. pylori.

In the present study, H. pylori eradication led to a significant reduction in the expression of p53. The number of p53-positive patients was decreased and the grade of positivity was also deceased. Kodama [1] also described the same observation. They reported that H. pylori eradication reduced gastritis activity, atrophy and complete metaplasia, accompanied by the disappearance of genomic instability markers.

After eradication therapy, the number of cases and the grade of positive c-Myc expression were significantly deceased similar to other reports [7] which suggest that H. pylori downregulates p27 and that this is reversed following H. pylori eradication.

Finally, we can conclude that H. pylori seems to be responsible for genomic instability and its eradication can reverse inflammation and related atrophy, metaplasia and genomic instability. Therefore, we recommend that H. pylori infected patients especially those with precancerous lesions should receive intense eradication therapy and should be closely monitored. Eradication reverses the atrophic changes in the gastric mucosa and the genetic instability and may, thus, prevent the development of gastric cancer.