Zhou X, Wang LQ, Song S, Xu M, Li CP. Helicobacter pylori infection promotes the progression of gastric cancer by regulating the expression of DMBT1. World J Clin Oncol 2025; 16(5): 105322 [DOI: 10.5306/wjco.v16.i5.105322]
Corresponding Author of This Article
Chang-Ping Li, MD, Department of Gastroenterology, The Affiliated Hospital of Southwest Medical University, No. 25 Taiping Street, Luzhou 646000, Sichuan Province, China. 506854209@qq.com
Research Domain of This Article
Gastroenterology & Hepatology
Article-Type of This Article
Basic Study
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Xiu Zhou, Lin-Qing Wang, Shuai Song, Mei Xu, Chang-Ping Li, Department of Gastroenterology, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
Author contributions: Zhou X and Wang LQ contributed equally as co-first authors; Zhou X designed the experimental plan, conducted relevant experiments, and wrote the original article; Wang LQ conducted relevant experiments, collected data and performed statistical analysis of data; Song S acquired materials, assisted in conducting experiments, and supplemented the article; Xu M assisted in completing experiments; Li CP supervised experiments and methods, and edited the article. All authors read and approved the final manuscript.
Institutional review board statement: The study was reviewed and approved by the Institutional Clinical Trial Ethics Committee of Affiliated Hospital of the Southwest Medical University (Approval No.KY2025054).
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: No additional data are available.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Chang-Ping Li, MD, Department of Gastroenterology, The Affiliated Hospital of Southwest Medical University, No. 25 Taiping Street, Luzhou 646000, Sichuan Province, China. 506854209@qq.com
Received: January 18, 2025 Revised: March 4, 2025 Accepted: March 26, 2025 Published online: May 24, 2025 Processing time: 122 Days and 2.9 Hours
Abstract
BACKGROUND
Each year, more than a million people are diagnosed with gastric cancer (GC) worldwide, and the incidence of this disease is projected to increase. Helicobacter pylori (H. pylori) is the major cause of GC. Managing infections caused by H. pylori and investigating their contribution to GC carcinogenesis are crucial for advancing diagnosis and treatment. Deleted in malignant brain tumors 1 (DMBT1) is associated with the development of H. pylori and GC. However, the precise underlying mechanism is unclear.
AIM
To explore the role of DMBT1, as modulated by H. pylori, in the development, proliferation, and metastasis of GC.
METHODS
Utilizing human GC cells, DMBT1 gene silencing, and H. pylori treatment, four cell groups (control, H. pylori, si-DMBT1, and H. pylori + si-DMBT1) were subjected to cell counting kit-8, scratch, and Transwell assays. The DMBT1 expression was assessed by quantitative real-time polymerase chain reaction and Western blot.
RESULTS
In cellular tests, H. pylori + si-DMBT1 showed the greatest ability to proliferate, migration, and invasion capabilities, followed by the si-DMBT1, H. pylori, and control groups. DMBT1 mRNA was found to be the highest in control group, next in si-DMBT1, H. pylori and H. pylori + si-DMBT1, while H. pylori + si-DMBT1 showed the least expression. The results the Western blot assay showed a consistent trend of decreasing DMBT1 protein and mRNA levels.
CONCLUSION
Through inhibition of DMBT1, H. pylori could enhance GC’s proliferation, metastasis and invasion. Our findings revealed a novel connection between H. pylori infection, inflammation, and GC.
Core Tip: The main reason for gastric cancer (GC) is Helicobacter pylori (H. pylori). The annual global burden of GC continues to increase. Our study investigated the influence of deleted in malignant brain tumors 1 induced by H. pylori on the proliferation and metastasis of GC. Using human GC cells, we concluded that H. pylori promote GC cell proliferation, metastasis, and invasion through down-regulation of deleted in malignant brain tumors 1 and facilitates GC development through cell function experiments and gene expression experiments.
Citation: Zhou X, Wang LQ, Song S, Xu M, Li CP. Helicobacter pylori infection promotes the progression of gastric cancer by regulating the expression of DMBT1. World J Clin Oncol 2025; 16(5): 105322
According to the 2022 global cancer statistics, gastric cancer (GC) ranks as the fifth most common and fifth most fatal cancer worldwide[1]. Millions of new cases are diagnosed annually, posing severe threats to human health. The Correa model depicts the progression of the stomach mucosa from the inflammation phase into the carcinoma phase, and identifies chronic atrophic gastritis, dysplasia, and intestinal metaplasia as premalignant diseases[2]. GC develops through complex mechanisms involving multiple genetic and environmental factors. Despite rapid advancements in treatment, the mortality rate remains high, underscoring the need for ongoing research on the mechanisms driving GC progression and the identification of effective therapeutic targets.
Helicobacter pylori (H. pylori), a gram negative bacteria, is found in the stomach lining of the stomach, causing inflammation, including gastritis, peptic ulceration and GC[3]. In 1994, H. pylori was formally designated by the World Health Organization as a category I carcinogen for GC[4]. Consensus guidelines on managing H. pylori infections recommend prompt eradication therapy upon detection to maximize health outcomes[5]. In addition, H. pylori has been recognized by the Maastricht VI Consensus as a major contributor to stomach adenocarcinoma[6]. Studies show that H. pylori promotes GC formation through a number of mechanisms; it triggers inflammatory stress reactions, utilizes the virulence factor CagA, and disrupts the critical balance between cell proliferation and apoptosis[7-9]. H. pylori secretes colonization- and pathogenesis-related molecules into the stomach[10,11]. It can counteract acid environment in stomach cavity, avoid host immunity reaction, build permanent infection, and induce chronic inflammation. This chronic state can evolve over several years into precancerous conditions, such as atrophic gastritis, intestinal metaplasia, and dysplasia[12], eventually leading to GC. Thus, it is important to know the molecular mechanism of H. pylori infection in patients with premalignant stomach disease and GC to find new treatment strategies for those diseases.
Deleted in malignant brain tumors 1 (DMBT1), which lies at the end of chromosome 10 (10q25.3-26.1), was initially recognized as a result of the absence of expression in malignant brain tumors[13]. DMBT1 encodes three types of secretory proteins and is a member of the cysteine-rich scavenger receptor superfamily[14]. Further research has also shown that DMBT1 has been lost in various cancers, including those of the gastrointestinal tract, lungs, breasts, and thyroid[15-17]. DMBT1 interacts with various bacteria, including H. pylori, contributing to the innate immune response[18]. In addition, DMBT1 regulates epithelial cell differentiation and affects the equilibrium between cell proliferation and apoptosis[19]. DMBT1’s diverse functions include acting as a tumor suppressor, regulating epithelial differentiation, contributing to innate immunity, and promoting angiogenesis[20-22]. Research on its role as a tumor suppressor is the most extensive, making it a current focus and hotspot. DMBT1 expression is significantly association with H. pylori and GC, and by combining in vitro experiments, we aimed to elucidate the interactions between H. pylori, DMBT1, and GC to provide insight into the development of GC treatments.
MATERIALS AND METHODS
H. pylori culture
The Third Military Medical University’s standard H. pylori strain SS1 was kept at -80 °C. Upon acclimatization at room temperature, the bacterium was seeded on a Columbia solid culture and incubated in an environment containing 5% O2, 10% CO2 and 85% N2 at 37 °C. After 48-72 hours of incubation, translucent, smooth, and pinpointed colonies were observed. Bacterial identification was confirmed by Gram staining, urease activity tests, and catalase tests.
Cell culture and transfection
The AGS human carcinoma cell line obtained from Saibai Kang Biotechnology Company was kept at -80 °C. Then, it was incubated in F-12 K culture medium with 10% fetal cow serum after defrosting, centrifugation and culture. Before transfection, the cells are grown on six well plates, and the transfection is started at about 40% convergence. Each well was supplemented with 500 μL of transfection mixture, composed of 250 μL Opti-MEM, 5 μL siRNA, and 5 μL Lipofectamine 2000, followed by thorough mixing. The cells were collected after 48 hours for further analysis. The siRNA sequence targeting DMBT1 employed was 5’-GCTGCAACTATGATTAUATdTdT-3’.
Co-cultivation of H. pylori with AGS cells
H. pylori was injected into the AGS cell culture plates at a density of 1.5 × 108 colony formation units per ml and incubated under the conditions of 5% O2, 10% CO2 and 85% N2 at 37 ºC. After incubation, the cells are collected for the purpose of obtaining RNA and protein for further testing.
Cell proliferation assay
Log-phase cells from each group (control, H. pylori, si-DMBT1, and H. pylori + si-DMBT1) were re-suspended at 1 × 105 cell/mL. Cells were seeded at a rate of 1 × 104 cells in 96 well plates, with 100 μl per well. Then, after 12 hours of hunger, the culture was changed to a special culture for every group, and then incubated for 24 hours, including the control and the blank. Then, each well is filled with a 10 mL cell counting kit-8 (CCK-8) solution, and the plates are incubated at 37 °C under 5% O2, 10% CO2 and 85% N2 for 1-4 hours. The absorbance was measured at 450 nm with a spectrophotometer. The experiment was repeated three times.
Cell migration test
Parallel lines were evenly drawn across the back of a six well plate using a marker. Then, the cells were seeded into wells, with each well containing 2 mL of a cell suspension containing approximately 5 × 105 cells. Once the cells had fully spread across the bottom of the wells, the tip of a pipette was held perpendicular to the pre-drawn lines to gently create a scratch and remove a strip of cells. Thereafter, a serum free medium is added and the plates are incubated at 37 °C with 5% O2, 10% CO2 and 85% N2 respectively. Cell migration was observed and documented photographically at 0 hour and 24 hours post-scratching. The experiment was repeated three times.
Cell invasion assay
Preparation of whole culture medium (500 μL) with 10% fetal cattle serum, and aliquoted in 24 well Transwell plate. Centrifuge a 1 × 105 cell/mL cell suspension for 5 minutes at 1500 revolutions per minute to secure a homogenous suspension, 200 μL was transferred onto the upper chamber of a Transwell insert. The cells were incubated for 48 hours at 37 °C. Following incubation, Transwell inserts are removed, phosphate buffered saline is used, and fixed for 20 minutes in polyformaldehyde. Following the second phosphate buffered saline flush, the cells were stained for 10 minutes at 25 °C with crystalline purple, then washed once more, and the unseeded surface was taken by reverse microscopy. The experiment was repeated three times.
Quantitative real-time polymerase chain reaction
Total RNA was extracted from AGS cells using TRIzol agent. According to the instruction provided by the EntiLink™ cDNA Synthesis Super Mix Kit (EQ031, ELK Biotechnology), cDNA was synthesized by reverse transcription. Quantitative real-time polymerase chain reaction (PCR) was performed using an EnTurbo ™ SYBR Green PCR SuperMix (EQ001, ELK Biotechnology). The experiment was repeated three times.
Western blot
The AGS cells were treated with a radio immunoprecipitation assay lysate buffer to extract the whole protein. The bicinchoninic acid protein test kit was used to determine the isolation protein levels, followed by sodium-dodecyl sulfate gel electrophoresis and transported on the membrane. The film was sealed for one hour at 25 °C, the blockage was removed, and then the dilution of the first antibody was administered and then incubated at 4 °C for one night, followed by the addition of the diluted second antibody and incubation at ambient temperature for 30 minutes. Optical densities of target bands were quantified with the AlphaEaseFC software system. The experiment was repeated three times.
Statistical analysis
Statistically analyzed with SPSS 27.0, and the graph is produced by GraphPad Prism 9.0. The data underwent normality testing and homogeneity of variance testing. The hypothesis that all groups were normal (P > 0.05) and analysis of variance was homogeneous (P > 0.05). Experimental results are presented as mean ± SD. One-way analysis of variance was applied to compare the results of the study. Statistical significance was observed at P < 0.05.
RESULTS
Influence of H. pylori on the expression of DMBT1 in GC
In order to clarify DMBT1’s function in H. pylori-caused GC, we employed gene silencing technology for and conducted co-culture experiments with H. pylori and AGS cells. The expression of DMBT1 in AGS cells decreased obviously when it was co-cultivated with H. pylori. A further reduction in DMBT1 expression was observed when AGS cells pre-silenced for DMBT1 were cocultured with H. pylori, indicating an additive effect (Figure 1, Table 1). Western blotting and quantitative real-time PCR analyses showed concordant trends in the reduction of DMBT1 protein and mRNA levels, respectively (Figure 2, Table 2). The results indicated that H. pylori could suppress DMBT1 expression in GC cells, so we conducted a functional cell biology experiment.
Figure 1 Quantitative real-time polymerase chain reaction to detect the relative expression of deleted in malignant brain tumors 1 mRNA in AGS cells of each group.bP < 0.001, cP < 0.0001. H. pylori: Helicobacter pylori; DMBT1: Deleted in malignant brain tumors 1.
Figure 2 Western blot detection of the relative expression of deleted in malignant brain tumors 1 mRNA in AGS cells of each group.aP < 0.01, bP < 0.001. H. pylori: Helicobacter pylori; DMBT1: Deleted in malignant brain tumors 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
Table 1 Comparison of relative expression of deleted in malignant brain tumors 1 mRNA in each group.
Influence of H. pylori infection on GC cell biology behavior
In the CCK-8, scratch, and Transwell assays, the groups treated with H. pylori + si-DMBT1 exhibited significant differences compared with the si-DMBT1, H. pylori group, and control groups. Cell treatment with both si-DMBT1 and H. pylori resulted in a significant increase in the proliferation rate (Figure 3A), migration (Figure 3B and C), and invasion (Figure 3D and E) measurements compared to application of si-DMBT1 or H. pylori alone (Tables 3, 4 and 5). These functional assay results are consistent with changes in DMBT1 expression levels, indicating that H. pylori-infected could increase the proliferation, migration and invasion of GC by reducing DMBT1.
Figure 3 Cell counting kit-8, scratch, and Transwell assays to detect the biological behavior in AGS cells of each group.
A: Cell counting kit-8 assay was employed to detect the proliferation ability of AGS cells in each group; B and C: Cell scratch assay to detect the migration ability of AGS cells in each group (40 ×); D and E: Transwell assay was performed in AGS cells to detect the invasion ability in each group (40 ×). bP < 0.001,cP < 0.0001. H. pylori: Helicobacter pylori; DMBT1: Deleted in malignant brain tumors 1; OD: Optical density.
Table 3 Comparison of relative proliferative capacities in each group.
While H. pylori incidence has decreased worldwide, it remains high, particularly in developing countries[23,24]. The failure to eradicate this H. pylori can significantly jeopardize human health, since H. pylori can catalyze GC initiation and progression through modulation of several oncogenes and tumor suppressor genes. In vitro studies showed that DMBT1 was inhibited by H. pylori-induced inhibition during GC progression. Decreased DMBT1 expression in H. pylori infected patients may promote the proliferation, metastasis and invasion of GC cells. Therefore, DMBT1 is critical for the progression of preneoplastic disease induced by H. pylori and its consequent GC formation.
DMBT1 is a multifunctional protein expressed in the gastrointestinal tract, nasal passages, lungs, thyroid, and other tissues[13,15,25,26], and plays a significant role in both inflammation and tumor processes. Specifically, which is responsible for inhibition of the progression of gall bladder carcinoma through phosphatidylinositol 3-kinase (PI3K)/AKT[27]. In another study, Ma and Zhao[28] demonstrated that DMBT1 can suppress the proliferation, migration and invasion of ovarian cancer through its galectin-3/PI3K/AKT pathway, thereby suppressing cancer progression and enhancing its chemotherapy sensitivity. Garay et al[29] investigated the pathologic alterations of the stomach mucous membrane in H. pylori deficient mice, which were deficient in DMBT1, and observed an increase in serious inflammation and widespread mucous metaplasia in comparison with those in the wild. The above findings suggest that DMBT1 may be involved in protecting stomach mucosa following H. pylori infection. The amount of DMBT1 in Iran’s saliva was much higher compared to that of normal people[30]. But recently, it has been shown that DMBT1 is down-regulated in GC cells. Wang et al[31] proposed that reg3A may be involved in inhibiting the growth of GC cells by targeting DMBT1.
In line with earlier research, DMBT1 mRNA and translation were markedly reduced in GC cells after H. pylori infected. Additionally, CCK-8 assays demonstrated an enhanced proliferative capacity of AGS cells post-infection, with increased migration and invasion abilities, supported by scratch and Transwell-experiments, respectively. This aligns with the finding that DMBT1 overexpression inhibits these oncogenic processes in cervical cancer cells[31]. Thus, it can reasonably be assumed that H. pylori induces a Correa’s cascade[32], which can facilitate the proliferation, migration and invasion of GC cells by downregulating DMBT1, which promotes GC initiation and development.
Research by Sugano et al[33] involving staining, sectioning, and mass spectrometry analysis of postsurgical GC tissues indicated upregulation of DMBT1 from normal gastric mucosa through metaplasia to cancer progression. Additionally, studies by Sousa et al[34] on gastric biopsy tissues from three different American populations revealed increased DMBT1 expression in conditions such as atrophic gastritis and intestinal metaplasia, indicating that DMBT1 up-regulation might begin at the beginning of the chain reaction and become more elevated in the course of progression. Generally speaking, the results of studies on DMBT1 expression in premalignant stomach disease and GC are different. These inconsistencies may be attributed to individual variability among samples, types of specimens, and differences in tumor stages and histopathological types. The specific interaction of H. pylori with GC was examined, as well as the role of DMBT1 in both diseases. What we have discovered is that down-regulating DMBT1 induced by H. pylori can enhance the proliferation, migration and invasion of GC cells. However, the precise molecular mechanisms underlying DMBT1’s role in this context remain to be elucidated. The next research will be aimed at defining DMBT1’s specific regulation mechanism, including its transcription and interaction with PI3K/AKT, Wnt/β Catenin. In order to confirm the results, we intend to build an in vivo H. pylori-infected rat model and carry out immunohistochemistry on GC biopsy samples to confirm the DMBT1 expression. The goal of this effort is to improve our knowledge of DMBT1’s regulation in the development of GC.
CONCLUSION
In conclusion, H. pylori inhibits DMBT1 to increase the proliferation, metastasis and invasion of GC cells. These insights provide new directions for understanding the molecular mechanisms underlying GC pathogenesis.
Footnotes
Provenance and peer review: Unsolicited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Oncology
Country of origin: China
Peer-review report’s classification
Scientific Quality: Grade D
Novelty: Grade D
Creativity or Innovation: Grade C
Scientific Significance: Grade C
P-Reviewer: Guo Z S-Editor: Wei YF L-Editor: A P-Editor: Zhao YQ
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