Edited by Xu XQ and Wang JH Verified by Ma JY
Published online Dec 15, 2001. doi: 10.3748/wjg.v7.i6.779
Revised: July 19, 2001
Accepted: August 20, 2001
Published online: December 15, 2001
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.
- Citation: Zhang Z, Yuan Y, Gao H, Dong M, Wang L, Gong YH. Apoptosis, proliferation and p53 gene expression of H. pylori associated gastric epithelial lesions. World J Gastroenterol 2001; 7(6): 779-782
- URL: https://www.wjgnet.com/1007-9327/full/v7/i6/779.htm
- DOI: https://dx.doi.org/10.3748/wjg.v7.i6.779
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 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.
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. 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.
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.
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.
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.
t test was used to compare the means. The positivity of p53 protein was analyzed by χ2 test
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.
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.
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.
|Group||n||H. pylori||n||p53 positive n (%)|
Gastric mucosa consists of continuously renewed cells and cell proliferation and apoptosis maintain their balance. 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, 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. 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; 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.
|1.||Wang TC, Fox JG. Helicobacter pylori and gastric cancer: Koch's postulates fulfilled. Gastroenterology. 1998;115:780-783. [PubMed] [DOI]|
|2.||Pan KF, Liu WD, Ma JL, Zhou T, Zhang L, Chang YS, You WC. Infection of He licobacter pylori in children and mode of transmission in a high risk area of ga stric cancer. Huaren Xiaohua Zazhi. 1998;6:42-44.|
|4.||Cai L, Yu SZ, Zhang ZF. Helicobacter pylori infection and risk of gastric cancer in Changle County, Fujian Province, China. World J Gastroenterol. 2000;6:374-376. [PubMed]|
|5.||Zhuang XQ, Lin SR. Research of Helicobacter pylori infection in precancerous gastric lesions. World J Gastroenterol. 2000;6:428-429. [PubMed]|
|6.||Iseki K, Tatsuta M, Iishi H, Baba M, Ishiguro S. Helicobacter pylori infection in patients with early gastric cancer by the endoscopic phenol red test. Gut. 1998;42:20-23. [PubMed] [DOI]|
|7.||Oderda G. Management of Helicobacter pylori infection in children. Gut. 1998;43 Suppl 1:S10-S13. [PubMed] [DOI]|
|8.||Liang YR, Zheng SY, Shen YQ, Wu XY, Huang ZZ. Relationship between expressi on of apoptosis related antigens in hepatocellular carcinoma and in situ end labeling. Huaren Xiaohua Zazhi. 1998;6:236-239.|
|9.||Luo HT, Chang WH, Ji XL. Light and electron microscopy study of Helicobacter pylori associated gastritis and gastric carcinoma. Huaren Xiaohua Zazhi. 1998;6:282-284.|
|10.||Hua JS, Zheng PY, Bow H. Species differentiation and identification in the genus of Helicobacter. World J Gastroenterol. 1999;5:7-9. [PubMed]|
|11.||Komoto K, Haruma K, Kamada T, Tanaka S, Yoshihara M, Sumii K, Kajiyama G, Talley NJ. Helicobacter pylori infection and gastric neoplasia: correlations with histological gastritis and tumor histology. Am J Gastroenterol. 1998;93:1271-1276. [PubMed] [DOI]|
|12.||Hua JS, Zheng PY, Bow H, Megraud F. Detection of Helicobacter pylori in gastric biopsy by polymerase chain reaction. Huaren Xiaohua Zazhi. 1998;6:377-379.|
|13.||Anti M, Armuzzi A, Gasbarrini A, Gasbarrini G. Importance of changes in epithelial cell turnover during Helicobacter pylori infection in gastric carcinogenesis. Gut. 1998;43 Suppl 1:S27-S32. [PubMed] [DOI]|
|14.||Ma J L, Liu WD, Zhang ZZ, Zhang L, You WC, Chang YS. Relationship between gastric cancer and precancerous lesions. Huaren Xiaohua Zazhi. 1998;6:222-223.|
|15.||Liu WZ, Zheng X, Shi Y, Dong QJ, Xiao SD. Effect of Helicobacter pylori infection on gastric epithelial proliferation in progression from normal mucosa to gastriccarcinoma. World J Gastroenterol. 1998;4:246-248. [PubMed]|
|16.||Badov D, Lambert JR, Finlay M, Balazs ND. Helicobacter pylori as a pathogenic factor in Ménétrier's disease. Am J Gastroenterol. 1998;93:1976-1979. [PubMed]|
|17.||Tucci A, Poli L, Tosetti C, Biasco G, Grigioni W, Varoli O, Mazzoni C, Paparo GF, Stanghellini V, Caletti G. Reversal of fundic atrophy after eradication of Helicobacter pylori. Am J Gastroenterol. 1998;93:1425-1431. [PubMed] [DOI]|
|18.||Lu W, Chen LY, Gong HS. PCNA and c-erbB-2 expression in gastric mucosal intestinal metaplasia with Helicobacter pylori infection. Shijie Huaren Xiaohua Zazhi. 1999;7:111-113.|
|19.||Wang YK, Ji XL, Ma NX. Expressi ons of p53 bcl-2 and c-erbB-2 proteins in precarcinomatous gastric mucosa. Shijie Huaren Xiaohua Zazhi. 1999;7:114-116.|
|20.||Hou P, Tu ZX, Xu GM, Gong YF, Ji XH, Li ZS. Helicobacter pylori vacA genotypes and cagA status and their relationship to associated diseases. World J Gastroenterol. 2000;6:605-607. [PubMed]|
|21.||Yang ZB, Wang PL, Gu MM, Chen LH, Chen Q, Zhan L. Diagnostic value of CagA IgG in the process to eradicateHelicobacter pylori. World J Gastroenterol. 2000;6:70.|
|22.||de Figueiredo Soares T, de Magalhães Queiroz DM, Mendes EN, Rocha GA, Rocha Oliveira AM, Alvares Cabral MM, de Oliveira CA. The interrelationship between Helicobacter pylori vacuolating cytotoxin and gastric carcinoma. Am J Gastroenterol. 1998;93:1841-1847. [PubMed] [DOI]|
|23.||Camorlinga-Ponce M, Torres J, Perez-Perez G, Leal-Herrera Y, Gonzalez-Ortiz B, Madrazo de la Garza A, Gomez A, Muñoz O. Validation of a serologic test for the diagnosis of Helicobacter pylori infection and the immune response to urease and CagA in children. Am J Gastroenterol. 1998;93:1264-1270. [PubMed]|
|24.||Basso D, Navaglia F, Brigato L, Piva MG, Toma A, Greco E, Di Mario F, Galeotti F, Roveroni G, Corsini A. Analysis of Helicobacter pylori vacA and cagA genotypes and serum antibody profile in benign and malignant gastroduodenal diseases. Gut. 1998;43:182-186. [PubMed] [DOI]|
|25.||Zhang ZW, Patchett SE, Perrett D, Katelaris PH, Domizio P, Farthing MJ. The relation between gastric vitamin C concentrations, mucosal histology, and CagA seropositivity in the human stomach. Gut. 1998;43:322-326. [PubMed] [DOI]|
|26.||Maeda S, Ogura K, Yoshida H, Kanai F, Ikenoue T, Kato N, Shiratori Y, Omata M. Major virulence factors, VacA and CagA, are commonly positive in Helicobacter pylori isolates in Japan. Gut. 1998;42:338-343. [PubMed] [DOI]|
|27.||Figura N, Vindigni C, Covacci A, Presenti L, Burroni D, Vernillo R, Banducci T, Roviello F, Marrelli D, Biscontri M. cagA positive and negative Helicobacter pylori strains are simultaneously present in the stomach of most patients with non-ulcer dyspepsia: relevance to histological damage. Gut. 1998;42:772-778. [PubMed] [DOI]|
|28.||Rudi J, Rudy A, Maiwald M, Kuck D, Sieg A, Stremmel W. Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and relationship to gastrointestinal diseases. Am J Gastroenterol. 1999;94:1525-1531. [PubMed] [DOI]|
|29.||Gao GL, Yang Y, Yang SF, He J. Gastric carcinogenesis and reactivity of microvessel induced by N methyl N nitroN nitrosoguanidine (MNNG) in rats. Huaren Xiaohua Zazhi. 1998;6:109-111.|
|30.||Zhang H, Ren XL, Yao XX. T limphocyte subsets, nitric oxide, hexosamine and Helicobacter pylori infection in patients with chronic gastric diseases. Shijie Huaren Xiaohua Zazhi. 1999;7:127-129.|
|31.||Sun GY Liu WW, Zhou ZQ, Fang DC, Men RP, Luo YH. Free radicals in development of experimental gastric carcinoma and precancerous lesions induced by N-methyl-N’nitro N nitrosoguanidine in rats. World J Gastroenterol. 1998;4:124.|
|32.||Peng X, Feng JB, Wang SL. Distribution of nitric oxide synthase in stomach wall in rats. World J Gastroenterol. 1999;5:92. [PubMed]|
|33.||Salzman AL, Eaves-Pyles T, Linn SC, Denenberg AG, Szabó C. Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells. Gastroenterology. 1998;114:93-102. [PubMed]|
|34.||Drake IM, Mapstone NP, Schorah CJ, White KL, Chalmers DM, Dixon MF, Axon AT. Reactive oxygen species activity and lipid peroxidation in Helicobacter pylori associated gastritis: relation to gastric mucosal ascorbic acid concentrations and effect of H pylori eradication. Gut. 1998;42:768-771. [PubMed] [DOI]|
|35.||Shibata J, Goto H, Arisawa T, Niwa Y, Hayakawa T, Nakayama A, Mori N. Regulation of tumour necrosis factor (TNF) induced apoptosis by soluble TNF receptors in Helicobacter pylori infection. Gut. 1999;45:24-31. [PubMed] [DOI]|
|36.||Farinati F, Cardin R, Degan P, Rugge M, Mario FD, Bonvicini P, Naccarato R. Oxidative DNA damage accumulation in gastric carcinogenesis. Gut. 1998;42:351-356. [PubMed] [DOI]|
|37.||Mao LZ, Wang SX, Ji WF, Ren JP, Du HZ, He RZ. Comparative studies on p53 and PCNA expressions in gastric carcinoma between young and aged patients. Huaren Xiaohua Zazhi. 1998;6:397-399.|
|38.||Wang XH, Zhang WD, Zhang YL, Zeng JZ, Sun Y. Relationship betwe en Hp infection and oncogene and tumor suppressor gene expressions in gastric cancer and precan cerosis. Huaren Xiaohua Zazhi. 1998;6:516-518.|
|39.||Sun YX, Chen CJ, Zhou HG, Shi YQ, Pan BR, Feng WY. Expression of c-myc and p53 in colorectal adenoma and adenocarcinoma. Huaren Xiaohua Zazhi. 1998;6:1054-1056.|
|40.||Xu QW, Li YS, Zhu HG. Relationship between expression p53 protein, PCNA and CEA in colorectal cancer and lymph node metastasis. World J Gastroenterol. 1998;4:218.|
|41.||Lin GY, Chen ZL, Lu CM, Li Y, Ping XJ, Huang R. Immunohistochemical study on p53, H-rasp21, c-erbB-2 protein and PCNA expression in HCC tissues of Han and minority ethnic patients. World J Gastroenterol. 2000;6:234-238. [PubMed]|
|42.||Zheng SX, Liu LJ, Shao YS, Zh eng QP, Ruan YB, Wu ZB. Relationship between ras p53 gene RNA and protein expressionand HCC metastasis. Huaren Xiaohua Zazhi. 1998;6:104-105.|
|43.||Guo CQ, Wang YP, Liu GY, Ma SW, Ding GY, Li JC. Study on Helicobacter pylori infection and -p53, c-erbB-2 gene expression in carcinogenesis of gastric mucosa. Shijie Huaren Xiaohua Zazhi. 1999;7:313-315.|
|44.||Qin LJ. In situ hybridization of p53 tum or suppressor gene in human gastric precancerous lesions and gastric cancer. Shijie Huaren Xiaohua Zazhi. 1999;7:494-497.|
|45.||Zhang ZW, Farthing MJ. Molecular mechanisms of H. pylori associated gastric carcinogenesis. World J Gastroenterol. 1999;5:369-374. [PubMed]|
|46.||Gao H, Wang JY, Shen XZ, Liu JJ. Effect of Helicobacter pylori infection on gastric epithelial cell proliferation. World J Gastroenterol. 2000;6:442-444. [PubMed]|
|47.||Zhang X, Geng M, Wang YJ, Cao YC. Express ion of epidermal growth factor receptor and proliferating cell nuclear antigen in esophageal carcinoma and precancerous lesions. Huaren Xiaohua Zazhi. 1998;6:229-230.|
|48.||Fang YW, Xu GM, Zhu RM, Zhang HF, Li ZS. Expression of epidermal grow th factor mRNA in primary pancreatic adenocarcinoma. Huaren Xiaohua Zazhi. 1998;6:256-257.|
|49.||Hao YR, Ding GY, Chen PY, Geng JH. Effect of EGF receptor expres sion on cell proliferation in colorectal cancer. Huaren Xiaohua Zazhi. 1998;6:313-314.|
|50.||Nie ZH, Zheng WY, Guo RB, Li J, Zhang M, Zhu Y. Effect of EGF on peptic ulcer: relationship between H. pylori and gastricmucosal inflammation. Huaren Xiaohua Zazhi. 1998;6:600-602.|
|51.||Wang LP, Yu JY, Deng YJ, Tian YW, Wu X, L iu G, Ding HY. Relationship between the expression of somatostatin and epiderm al growth factor receptor in gastric carcinoma. Huaren Xiaohua Zazhi. 1998;6:60 6-609.|
|52.||Wang SM, Wu JS, Yao X, He ZS, Pan BR. Effect of TGF-α, EGFR antise nse oligodeoxynucleotides on colon cancer cell line. Shijie Huaren Xiaohua Zazhi. 1999;7:522-524.|