Apoptosis, proliferation and p53 gene expression of H. pylori associated gastric epithelial lesions

Abstract

AIM: To study the relationship between Helicobacter pylori (H. pylori) and gastric carcinoma and its possible pathogenesis by H. pylori.

METHODS: DNEL technique and immunohistochemical technique were used to study the state of apoptosis, proliferation and p53 gene expression. A total of 100 gastric mucosal biopsy specimens, including 20 normal mucosa, 30 H. pylori-negative and 30 H. pylori-positive gastric precancerous lesions along with 20 gastric carcinomas were studied.

RESULTS: There were several apoptotic cells in the superficial epithelium and a few proliferative cells within the neck of gastric glands, and no p53 protein expression in normal mucosa. In gastric carcinoma, there were few apoptotic cells, while there were a large number of proliferative cells, and expression of p53 protein significantly was increased. In the phase of metaplasia, the apoptotic index (AI, 4.36% ± 1.95%), proliferative index (PI, 19.11% ± 6. 79%) and positivity of p53 expression (46.7%) in H. pylori-positive group were higher than those in normal mucosa (P < 0.01). AI in H. pylori-positive group was higher than that in H. pylori-negative group (3.81% ± 1.76%), PI in H. pylori-positive group was higher than that in H. pylori-negative group (12.25% ± 5.63%, P < 0.01). In the phase of dysplasia, AI (2.31% ± 1.10%) in H. pylori-positive group was lower (3.05% ± 1.29%) than that in H. pylori-negative group, but PI (33.89% ± 11.65%) was significantly higher (22.09% ± 80.18%, P < 0.01). In phases of metaplasia, dysplasia and gastric cancer in the H. pylori-positive group, AIs had an evidently graduall decreasing trend (P < 0.01), while PIs had an evidently gradual increasing trend (P < 0.05 or P < 0.01), and there was also a trend of gradual increase in the expression of p53 gene.

CONCLUSION: In the course of the formation of gastric carcinoma, proliferation of gastric mucosa can be greatly increased by H. pylori, and H. pylori can induce apoptosis in the phase of metaplasia, but in the phase of dysplasia H. pylori can inhibit cellular apoptosis. And H. pylori infection can strengthen the expression of mutated p53 gene.

INTRODUCTION

H. pylori infection is epidemiologically associated with the development of gastric cancer[1-7], but it is unknown how H. pylori does so. In this study, DNEL technique[8] and immunohistochemical staining were used to dynamically observe and compare the state of apoptosis, proliferation and p53 gene expression in H. pylori-negative or H. pylori-positive gastric precancerous lesion as well as gastric carcinomas. The purpose is to probe into the effect of H. pylori on apoptosis, proliferation and p53 gene expression in gastric epithelium and to find out the relationship between H. pylori and gastric carcinogenesis, and the possible mechanism.

MATERIALS AND METHODS

Subjects

All samples were selected from people screened by endoscopy in a high risk area of gastric carcinoma in Zhuanghe, Liaoning Province. A total of 100 gastric mucosal biopsy specimens, including 20 normal mucosa, 30 metaplasia, 30 dysplasia and 20 gastric carcinoma cases. H. pylori infection was assessed by hematoxylin-eosin staining[9-11] and PCR[12]. If both results of the tests in a patient were positive, the patient was considered to be infected by H. pylori; if neither was positive, the patient was considered negative.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling method

Using the kit (Oncor, San Diego, USA), the staining steps are as follows: ① the sections were deparaffinised through xylem and alcohol, and washed;② digested for 15 min with proteinase K (20 mg•L¯¹); ③ quenched with 30 ML•L¯¹ H₂O₂ for 20 min; ④ applied with equilibration buffer for 15 s at room temperature; ⑤ added with terminal deoxynucleotidyl transferase (TdT) and incubated at 37 °C for 60 min; ⑥ added with STOP/WASF buffer at 37 °C for 10 min; ⑦ dropped with hydrogen peroxidase for 30 min at room temperature; and ⑧ visualized by immersi on in 3,3’-diaminobenzidine (DAB) solution, restained with methyl-green, and dehydrated, transparency, mounted. PBS was substituted for TdT as negative control.

Immunohistochemical staining

SP kit was used (Zymed, USA).The primary antibodies were PCNA monoclonal antibody (diluted 1:50) and p53 monoclonal antibody (ready to use, Maixin, Fijian), respectively. Before staining, the sections were microwave heated in 0.05 mol•L¯¹ citric acid solution for antigen retrieval. PBS was substituted for primary antibodies as negative control.

Observed parameters

Two samples were stained by DNEL and immunohistochemical staining, then the DNEL-positive cells (apoptotic cell) and PCNA- positive cells (proliferative cell) were observed. Apoptotic index (AI) and proliferative index (PI) were obtained by calculating the percentage of positively stained cells evaluated for each tissue section after counting 1000 cells at more than 5 high power fields.

Statistical analysis

t test was used to compare the means. The positivity of p53 protein was analyzed by χ² test

RESULTS

Charateristics of DNEL-positive, PCNA-positive and p53 protein-positive cells under microscopy

DNEL-positive cells (apoptotic cell) appeared brown corpuscular or diffuse in cell nuclei, and individual thickening nuclear membrane appeared brown. PCNA-positive cells (proliferative cell) appeared brown corpuscular in cell nuclei. Positive product of p53 expression was restricted in cell nuclei. In normal mucosa, apoptotic cells sporadically scattered on the epithelium, a few proliferative cells scattered on the glandular neck and expression of p53 protein was not seen. But in the tissue of gastric carcinoma, apoptotic cells accounted for 1.62%；proliferative cells for 41.99% and a cluster was formed all over the lesions; and there was a significant increase in the expression of p53 protein. Effect of H. pylori infection on apoptosis in gastric epithelium In the metaplasia mucosa, the apoptotic index in H. pylori-positive group was higher than that in normal mucosa (P < 0.01), and it was also higher than that in H. pylori-negative group; while in the dysplasia mucosa, AI in H. pylori-positive group was lower than that in H. pylori-negative group. In the metaplasia, dysplasia mucosa and gastric carcinoma AI presented with an evidently gradual decrease trend (P < 0.05 or P < 0.01 Table 1) in H. pylori-positive group.

Table 1.

Effect of H. pylori infection on AI in gastric epithelium (%, mean ± SD)

Group	H. pylori positive		H. pylori negative
	n	AI	n	AI
Normal	-	-	20	2.08 ± 1.07
Metaplasia	15	4.36 ± 1.95d	15	3.81 ± 1.76
Dysplasia	15	2.31 ± 1.10b	15	3.05 ± 1.29
Carcinoma	10	1.34 ± 0.69a	10	1.89 ± 1.03

^d P < 0.01, vs normal (H. pylori negative);

^a P < 0.05,

^bP < 0.01, vs upper adjacent group.

Effect of H. pylori infection on proliferation in gastric epithelium

In metaplasia and dysplasia mucosa the PI was significant higher than those in normal mucosa (P < 0.01), and that in H. pylori-positive group was higher than that in H. pylori-negative group (P < 0.01). From the normal mucosa to the gastric carcinoma, the PI has a gradual increase trend (P < 0.05 or P < 0.01, Table 2) in H. pylori-positive group.

Table 2.

Effect of H. pylori infection on PI in gastric epit helium (%, mean ± SD)

Group	H. pylori positive		H. pylori negative
	n	PI	n	PI
Normal	-	-	20	9.78 ± 3.65
Metaplasia	15	19.11 ± 6.79e	15	12.25 ± 5.63d
Dysplasia	15	33.89 ± 11.65eb	15	22.09 ± 8.18d
Carcinoma	10	48.27 ± 15.67ea	10	34.70 ± 12.74c

^eP < 0.01, vs normal (H. pylori negative);

^aP < 0.05,

^bP < 0.01, vs upper adjacent group;

^cP < 0.05,

^dP < 0.01, vs positive group.

Effect of H. pylori infection on the expression of p53 in gastric epithelium

In the metaplasia mucosa, the positivity of p53 protein expresion in H. pylori-positive group was higher than that in noraml mucosa (P < 0.01). In the metaplasia, dysplasia mucosa and gastric carcinoma, there was a trend of gradual increase in positivity of p53 protein expression (Table 3) in H. pylori-positive group.

Table 3.

Effect of H. pylori infecion on expression of p53 protein in gastric epithelium

Group	n	H. pylori	n	p53 positive n (%)
Normal	20		20	0 (0.0)
Metaplasia	30	+	15	7 (46.7)b
		-	15	1 (6.7)
Dysplasia	30	+	15	8 (53.3)
		-	15	5 (33.3)
Carcinoma	20	+	10	8 (80.0)
		-	10	7 (70.0)

^bP < 0.01, vs normal (H. pylori negative).

DISCUSSION

Gastric mucosa consists of continuously renewed cells and cell proliferation and apoptosis maintain their balance[13]. This study shows that the apoptotic cells were identified in gastric surface epithelium and formed “an apoptotic zone”; proliferative cells were seen in the neck region of the mucosal glands and formed “a prolife rating zone”. This distribution shows the proliferating zone gradually maturation, aging and death to the surface in the gastric mucosal epithelium. However in gastric carcinoma, apoptotic cells amount to 1.62%, proliferative cells amount to 41.99%, which clustered all over the tumor tissue. This change obviously lost the distribution characteristics of apoptotic zone and proliferating zone which elucidates that the regulation of apoptosis and proliferation have already been beyond the normal mucosa and appear significantly disordered in gastric carcinoma.

Human gastric carcinogenesis is a multistep and multifactorial process[14-17]. In this process, the state of apoptosis and proliferation of gastric epithelium will change[18,19]. In this study, H. pylori infection was found to affect the cell apoptosis and proliferation. In the metaplasia mucosa, AI was higher than that in normal mucosa in H. pylori-positive group, and higher than that in H. pylori-negative one; however, in the dysphasia mucosa, AI in H. pylori-positive group is lower than that in H. pylori-negative group. In the process of gastric carcinogenesis[14,15], from the phase of metaplasia, dysplasia to gastric carcinoma, AI gradually decreased in H. pylori-positive group. This shows that from normal mucosa to gastric carcinoma, H. pylori may induce cell apoptosis in the phase of metaplasia; but it inhibits cell apoptosis in the phase of dysplasia, this is familiar with gastric carcinoma. Several reports suggest that H. pylori produces cytotixic protein (CagA and VacA)[20-28], and gastric mucosa can increasex some cytokines, nitrous oxide sythetase and oxygen radicals released after H. pylori infection[29-35]. At the same time, H. pylori infection can lower the gastric antioxidant ability. All those factors make DNA dam age or enhance the susceptibility of DNA-damage, and those DNA-damaged cellscan be cleared away by apoptosis[36], and this may be the mechanism that H. pylori in duces apoptosis. It has been proved that wild type p53 protein can induce cell apoptosis but the intracellular accumulation of mutant p53 protein can inhibit cell apoptosis and promote cell transformation and proliferation, resulting in carcinogenesis[37-41]. In this study, in the phae of metaplasia, positivity of p53 protein expression in H. pylori-positive group was higher than that in normal mucosa. From the phase of metaplasia to gastric carcinoma, the positivity of p53 protein expression increased in H. pylori-positive group. As positive p53 protein confirmed by immunohistochemical staining was considered as mutation type[42]. It suggests that beginning with metaplasia, H. pylori infection can strengthen the expression of mutant p53 gene. With the accumulation of mutant and expression of p53, its inhibiting effect on apoptosis[43-45] will overpass the indution effect of H. pylori. Therefore in the H. pylori-positive dysplasia mucosa, there was a decrease in apoptosis. In this study, in the H. pylori-positive gastric precancerous lesions-metaplasia and dysplasia, PI was significantly higher than that in normal mucosa, and higher than those in the corresponding negative lesions. In the progress from normal mucosa to the malignant phenotype, PI gradually increased in H. pylori-positive group. The result shows that H. pylori apparently improves gastric epithelium proliferation which is very similar with the proliferating characteristics of gastric carcinoma. The mechanism may be that urease enzyme produced by H. pylori hydrolysesurea into carbon dioxide and ammonia, the latter can promote mitosis[46]; H. pylori infection will increase mucosal content and expression of EGF and TGF- α[47-52] and the effect of mutant p53 on proliferation, will result in rapid proliferation of epithelium.

In the phase of metaplasia, H. pylori can induce apoptosis, stimulate hyperproliferation and result in the regulation disorder of apoptosis and proliferation in the gastric epithelial cells, which increase the instability of gastric mucosa and carcinoma variability. Accompanied with the progress of lesions and the accumulation of p53 protein, H. pylori induces gastric epithelial cell hyperproliferation and apoptosis reduction or even imbalance of the apoptotic and proliferative process, and accumulation of DNA-damaged cells, ultimately resulting in gastric carcinogenesis. This makes it clear that H. pylori infection may be an important factor of gastric carcinogenesis. Thus, it is significant to prevent H. pylori infection, eradicate H. pylori in early stage and study the relationship between H. pylori infection and gastric carcinoma in order to decrease the incidence rate and prevent gastric carcinoma.