A single-nucleotide polymorphism in Helicobacter pylori promotes gastric cancer development.

Single-nucleotide polymorphisms (SNPs) in various human genes are key factors in carcinogenesis. However, whether SNPs in bacterial pathogens are similarly crucial in cancer development is unknown. Here, we analyzed 1,043 genomes of the stomach pathogen Helicobacter pylori and pinpointed a SNP in the serine protease HtrA (position serine/leucine 171) that significantly correlates with gastric cancer. Our functional studies reveal that the 171S-to-171L mutation triggers HtrA trimer formation and enhances proteolytic activity and cleavage of epithelial junction proteins occludin and tumor-suppressor E-cadherin. 171L-type HtrA, but not 171S-HtrA-possessing H. pylori, inflicts severe epithelial damage, enhances injection of oncoprotein CagA into epithelial cells, increases NF-κB-mediated inflammation and cell proliferation through nuclear accumulation of ß-catenin, and promotes host DNA double-strand breaks, collectively triggering malignant changes. These findings highlight the 171S/L HtrA mutation as a unique bacterial cancer-associated SNP and as a potential biomarker for risk predictions in H. pylori infections.

Genetic determinants of Biofilm formation of Helicobacter pylori using whole-genome sequencing.

BACKGROUND: Infection with Helicobacter pylori as the cause of gastric cancer is a global public health concern. In addition to protecting germs from antibiotics, biofilms reduce the efficacy of H. pylori eradication therapy. The nucleotide polymorphisms (SNPs) related with the biofilm forming phenotype of Helicobacter pylori were studied. RESULTS: Fifty-six H. pylori isolate from Bangladeshi patients were included in this cross-sectional study. Crystal violet assay was used to quantify biofilm amount, and the strains were classified into high- and low-biofilm formers As a result, strains were classified as 19.6% high- and 81.4% low-biofilm formers. These phenotypes were not related to specific clades in the phylogenetic analysis. The accessories genes associated with biofilm from whole-genome sequences were extracted and analysed, and SNPs among the previously reported biofilm-related genes were analysed. Biofilm formation was significantly associated with SNPs of alpA, alpB, cagE, cgt, csd4, csd5, futB, gluP, homD, and murF (P < 0.05). Among the SNPs reported in alpB, strains encoding the N156K, G160S, and A223V mutations were high-biofilm formers. CONCLUSIONS: This study revealed the potential role of SNPs in biofilm formation and proposed a method to detect mutation in biofilm from whole-genome sequences.

Comparative genomics of two Vietnamese Helicobacter pylori strains, CHC155 from a non-cardia gastric cancer patient and VN1291 from a duodenal ulcer patient.

Helicobacter pylori is involved in the etiology and severity of several gastroduodenal diseases; however, plasticity of the H. pylori genome makes complete genome assembly difficult. We report here the full genomes of H. pylori strains CHC155 and VN1291 isolated from a non-cardia gastric cancer patient and a duodenal ulcer patient, respectively, and their virulence demonstrated by in vitro infection. Whole-genome sequences were obtained by combining long- and short-reads with a hybrid-assembly approach. Both CHC155 and VN1291 genome possessed four kinds of genomic island: a cag pathogenicity island (cagPAI), two type 4 secretion system islands within an integrative and conjugative element (tfs ICE), and prophage. CHC155 and VN1291 carried East Asian-type cagA and vacA s1m1, and outer membrane protein genes, including two copies of oipA. Corresponded to genetic determinants of antibiotic resistance, chromosomal mutations were identified in CHC155 (rdxA, gyrA, and 23S rRNA) and VN1291 (rdxA, 23S rRNA, and pbp1A). In vitro infection of AGS cells by both strains induced the cell scattering phenotype, tyrosine phosphorylation of CagA, and promoted high levels of IL8 secretion, indicating fully intact phenotypes of the cagPAI. Virulence genes in CHC155 and VN1291 genomes are crucial for H. pylori pathogenesis and are risk factors in the development of gastric cancer and duodenal ulcer. Our in vitro studies indicate that the strains CHC155 and VN1291 carry the pathogenic potential.

Surfactin induces autophagy, apoptosis, and cell cycle arrest in human oral squamous cell carcinoma.

OBJECTIVES: To investigate the anticancer effects and underlying mechanisms of surfactin on human oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: The capacity of surfactin to induce apoptosis, autophagy, and cell cycle arrest of two different human OSCC cell lines was investigated by cell viability, acridine orange staining, and cell cycle regulatory protein expression, respectively. The signaling network underlying these processes were determined by the analysis of reactive oxygen species (ROS) generation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, endoplasmic reticulum (ER) stress-related protein levels, calcium release, mitogen-activated protein kinases activation, and cell cycle regulatory protein expression through corresponding reagents and experiments under various experimental conditions using specific pharmaceutical inhibitors or small interfering RNAs. RESULTS: Surfactin was able to induce apoptosis through NADPH oxidase/ROS/ER stress/calcium-downregulated extracellular signal-regulated kinases 1/2 pathway. Surfactin could also lead to autophagy that shared the common regulatory signals with apoptosis pathway until calcium node. Cell cycle arrest at G2 /M phase caused by surfactin was demonstrated through p53 and p21 accumulation combined p34cdc2 , phosphorylated p34cdc2 , and cyclin B1 inhibition, which was regulated by NADPH oxidase-derived ROS. CONCLUSION: Surfactin could induce apoptosis, autophagy, and cell cycle arrest in ROS-dependent manner, suggesting a multifaced anticancer agent for OSCC.

Genome-wide association study of gastric cancer- and duodenal ulcer-derived Helicobacter pylori strains reveals discriminatory genetic variations and novel oncoprotein candidates.

Genome-wide association studies (GWASs) can reveal genetic variations associated with a phenotype in the absence of any hypothesis of candidate genes. The problem of false-positive sites linked with the responsible site might be bypassed in bacteria with a high homologous recombination rate, such as Helicobacter pylori, which causes gastric cancer. We conducted a small-sample GWAS (125 gastric cancer cases and 115 controls) followed by prediction of gastric cancer and control (duodenal ulcer) H. pylori strains. We identified 11 single nucleotide polymorphisms (eight amino acid changes) and three DNA motifs that, combined, allowed effective disease discrimination. They were often informative of the underlying molecular mechanisms, such as electric charge alteration at the ligand-binding pocket, alteration in subunit interaction, and mode-switching of DNA methylation. We also identified three novel virulence factors/oncoprotein candidates. These results provide both defined targets for further informatic and experimental analyses to gain insights into gastric cancer pathogenesis and a basis for identifying a set of biomarkers for distinguishing these H. pylori-related diseases.

Surfactin attenuates particulate matter-induced COX-2-dependent PGE2 production in human gingival fibroblasts by inhibiting TLR2 and TLR4/MyD88/NADPH oxidase/ROS/PI3K/Akt/NF-κB signaling pathway.

OBJECTIVE: To evaluate the anti-inflammatory effects of surfactin and underlying mechanisms against particulate matter (PM)-induced inflammatory responses in human gingival fibroblasts (HGFs). BACKGROUND: PM, a major air pollutant, may associate with certain oral diseases possibly by inducing inflammation and oxidative stress. Surfactin, a potent biosurfactant, possesses various biological properties including anti-inflammatory activity. However, the underlying mechanisms are unclear. Also, there is no study investigating the effects of surfactin on PM-induced oral inflammatory responses. As an essential constituent of human periodontal connective tissues which involves immune-inflammatory responses, HGFs serve as useful study models. METHODS: HGFs were pretreated with surfactin prior to PM incubation. The PGE2 production was determined by ELISA, while the protein expression and mRNA levels of COX-2 and upstream regulators were measured using Western blot and real-time PCR, respectively. The transcriptional activity of COX-2 and NF-κB were determined using promoter assay. ROS generation and NADPH oxidase activity were identified by specific assays. Co-immunoprecipitation assay, pharmacologic inhibitors, and siRNA transfection were applied to explore the interplay of molecules. Mice were given one dose of surfactin or different pharmacologic inhibitors, then PM was delivered into the gingiva for three consecutive days. Gingival tissues were obtained for analyzing COX-2 expression. RESULTS: PM-treated HGFs released significantly higher COX-2-dependent PGE2 , which were regulated by TLR2 and TLR4/MyD88/NADPH oxidase/ROS/PI3K/Akt/NF-κB pathway. PM-induced COX-2/PGE2 increase was effectively reversed by surfactin through the disruption of regulatory pathway. Similar inhibitory effects of surfactin was observed in mice. CONCLUSION: Surfactin may elicit anti-inflammatory effects against PM-induced oral inflammatory responses.

The potentials of carbon monoxide-releasing molecules in cancer treatment: An outlook from ROS biology and medicine.

Carbon monoxide (CO) is now well recognized a pivotal endogenous signaling molecule in mammalian lives. The proof-of-concept employing chemical carriers of exogenous CO as prodrugs for CO release, also known as CO-releasing molecules (CO-RMs), has been appreciated. The major advantage of CO-RMs is that they are able to deliver CO to the target sites in a controlled manner. There is an increasing body of experimental studies suggesting the therapeutic potentials of CO and CO-RMs in different cancer models. This review firstly presents a short but crucial view concerning the characteristics of CO and CO-RMs. Then, the anticancer activities of CO-RMs that target many cancer hallmarks, mainly proliferation, apoptosis, angiogenesis, and invasion and metastasis, are discussed. However, their anticancer activities are varying and cell-type specific. The aerobic metabolism of molecular oxygen inevitably generates various oxygen-containing reactive metabolites termed reactive oxygen species (ROS) which play important roles in both physiology and pathophysiology. Although ROS act as a double-edged sword in cancer, both sides of which may potentially have been exploited for therapeutic benefits. The main focus of the present review is thus to identify the possible signaling network by which CO-RMs can exert their anticancer actions, where ROS play the central role. Another important issue concerning the potential effect of CO-RMs on the aerobic glycolysis (the Warburg effect) which is a feature of cancer metabolic reprogramming is given before the conclusion with future prospects on the challenges of developing CO-RMs into clinically pharmaceutical candidates in cancer therapy.

Pepsinogen and Serum IgG Detection Is a Valuable Diagnostic Method for Helicobacter pylori Infection in a Low-Prevalence Country: A Report from Sri Lanka.

The use of serum anti-Helicobacter pylori IgG and pepsinogen (PG) detection as a diagnostic method was evaluated in Sri Lanka. Gastric biopsies were performed (353 patients), and the prevalence of H. pylori infection was 1.7% (culture) and 2.0% (histology). IgG serology testing showed an area under the curve (AUC) of 0.922 (cut-off, 2.95 U/mL; specificity, 91.56%; sensitivity, 88.89%). Histological evaluation showed mild atrophy (34.3%), moderate atrophy (1.7%), metaplasia (1.7%), chronic gastritis (6.2%), and normal tissue (56%). The PGI/PGII ratio was significantly higher in H. pylori-negative patients (p < 0.01). PGII and PGI/PGII levels were lower in patients with metaplasia than in those with normal mucosa (p = 0.049 and p < 0.001, respectively). The PGI/PGII ratio best discriminated metaplasia and moderate atrophy (AUC 0.88 and 0.76, respectively). PGI and PGII alone showed poor discriminative ability, especially in mild atrophy (0.55 and 0.53, respectively) and chronic gastritis (0.55 and 0.53, respectively). The best cut-off to discriminate metaplasia was 3.25 U/mL (95.19% specificity, 83.33% sensitivity). Anti-H. pylori IgG and PG assessment (ABC method) was performed (group B, 2.0%; group A, 92.1%). The new cut-off more accurately identified patients with metaplasia requiring follow-up (group B, 5.4%). Assessment of anti-H. pylori IgG and PG is valuable in countries with a low prevalence of H. pylori infection.

Helicobacter pylori type 4 secretion systems as gastroduodenal disease markers.

Although the type 4 secretion system of the integrating and conjugative elements (tfs ICE) is common in Helicobacter pylori, its clinical association with the cag pathogenicity island (cagPAI) have not yet been well-investigated. In this study, Vietnamese patient H. pylori samples (46 duodenal ulcer (DU), 51 non-cardia gastric cancer (NCGC), 39 chronic gastritis (CG)) were fully sequenced using next-generation sequencing and assembled into contigs. tfs3, tfs4, and cagPAI genes were compared with the public database. Most (94%) H. pylori strains possessed a complete cagPAI, which was the greatest risk factor for clinical outcomes, while the prevalences of tfs3 and tfs4 were 45% and 77%, respectively. Complete tfs3 and tfs4 were found in 18.3% and 17.6% of strains, respectively. The prevalence of H. pylori strains with complete tfs3 ICE in DU patients was significantly higher than that in NCGC patients (30.4% vs 11.7%, P < 0.05). In addition, the prevalence of strains with complete tfs3 ICE and cagPAI was significantly higher in DU patients than that in NCGC (28.4% vs 9.8%, P = 0.038) and CG patients (28.2% vs 7.7%, P = 0.024). cagPAI and complete tfs3 increased the risk of DU compared to NCGC (OR = 3.56, 95%CI: 1.1-14.1, P = 0.038) and CG (OR = 4.64, 95%CI: 1.1-27.6, P = 0.024). A complete cluster of tfs3 ICE was associated with gastroduodenal diseases in Vietnam. However, there was a low prevalence of the dupA/complete dupA cluster (15.4%) in the Vietnam strains. The prevalence of cagPAI in Vietnam strains was significantly higher than in US (P = 0.01) and Indonesia (P < 0.0001); the prevalence of the dupA cluster was also higher in the Vietnam strains than in the Indonesian strains (P < 0.05). In addition, the prevalence of ctkA, an accessory gene of tfs3, was significantly different between Vietnam and US strains (28% vs 2%, P = 0.0002). In summary, the acquisition of tfs3/4 ICE was common in H. pylori strains in patients with gastroduodenal disease in Vietnam, and the complete cluster of tfs3 ICE was a reliable marker for the severity of disease in the H. pylori infected population.

Serum Helicobacter pylori antibody reactivity in seven Asian countries using an automated latex aggregation turbidity assay.

BACKGROUND AND AIM: To determine the application range of diagnostic kits utilizing anti-Helicobacter pylori antibody, we tested a newly developed latex aggregation turbidity assay (latex) and a conventional enzyme-linked immunosorbent assay (E-plate), both containing Japanese H. pylori protein lysates as antigens, using sera from seven Asian countries. METHODS: Serum samples (1797) were obtained, and standard H. pylori infection status and atrophy status were determined by culture and histology (immunohistochemistry) using gastric biopsy samples from the same individuals. The two tests (enzyme-linked immunosorbent assay and latex) were applied, and receiver operating characteristics analysis was performed. RESULTS: Area under the curve (AUC) from the receiver operating characteristic of E-plate and latex curves were almost the same and the highest in Vietnam. The latex AUC was slightly lower than the E-plate AUC in other countries, and the difference became statistically significant in Myanmar and then Bangladesh as the lowest. To consider past infection cases, atrophy was additionally evaluated. Most of the AUCs decreased using this atrophy-evaluated status; however, the difference between the two kits was not significant in each country, but the latex AUC was better using all samples. Practical cut-off values were 3.0 U/mL in the E-test and 3.5 U/mL in the latex test, to avoid missing gastric cancer patients to the greatest extent possible. CONCLUSIONS: The kits were applicable in all countries, but new kits using regional H. pylori strains are recommended for Myanmar and Bangladesh. Use of a cut-off value lower than the best cut-off value is essential for screening gastric cancer patients.

Diffuse Gastric Cancer: A Summary of Analogous Contributing Factors for Its Molecular Pathogenicity.

Gastric cancer is the third leading cause of cancer-related deaths and ranks as the fifth most common cancer worldwide. Incidence and mortality differ depending on the geographical region and gastric cancer ranks first in East Asian countries. Although genetic factors, gastric environment, and Helicobacter pylori infection have been associated with the pathogenicity and development of intestinal-type gastric cancer that follows the Correa's cascade, the pathogenicity of diffuse-type gastric cancer remains mostly unknown and undefined. However, genetic abnormalities in the cell adherence factors, such as E-cadherin and cellular activities that cause impaired cell integrity and physiology, have been documented as contributing factors. In recent years, H. pylori infection has been also associated with the development of diffuse-type gastric cancer. Therefore, in this report, we discuss the host factors as well as the bacterial factors that have been reported as associated factors contributing to the development of diffuse-type gastric cancer.

Molecular Epidemiology of Helicobacter pylori Infection in a Minor Ethnic Group of Vietnam: A Multiethnic, Population-Based Study.

The Helicobacter pylori-induced burden of gastric cancer varies based on geographical regions and ethnic grouping. Vietnam is a multiethnic country with the highest incidence of gastric cancer in Southeast Asia, but previous studies focused only on the Kinh ethnic group. A population-based cross-sectional study was conducted using 494 volunteers (18-78 years old), from 13 ethnic groups in Daklak and Lao Cai provinces, Vietnam. H. pylori status was determined by multiple tests (rapid urease test, culture, histology, and serology). cagA and vacA genotypes were determined by PCR-based sequencing. The overall H. pylori infection rate was 38.1%. Multivariate analysis showed that variations in geographical region, age, and ethnicity were independent factors associated with the risk of H. pylori acquisition. Therefore, multicenter, multiethnic, population based study is essential to assess the H. pylori prevalence and its burden in the general population. Only the E De ethnicity carried strains with Western-type CagA (82%) and exhibited significantly lower gastric mucosal inflammation compared to other ethnic groups. However, the histological scores of Western-type CagA and East-Asian-type CagA within the E De group showed no significant differences. Thus, in addition to bacterial virulence factors, host factors are likely to be important determinants for gastric mucosal inflammation and contribute to the Asian enigma.

Genetic populations and virulence factors of Helicobacter pylori.

Helicobacter pylori is a bacterium that has infected more than half of the human population worldwide. This bacterium is closely associated with serious human diseases, such as gastric cancer, and identifying and understanding factors that predict bacterial virulence is a priority. In addition, this pathogen shows high genetic diversity and co-evolution with human hosts. H. pylori population genetics, therefore, has emerged as a tool to track human demographic history. As the number of genome sequences available is increasing, studies on the evolution and virulence of H. pylori are gaining momentum. This review article summarizes the most recent findings on H. pylori virulence factors and population genetics.

Advanced non-cardia gastric cancer and Helicobacter pylori infection in Vietnam.

BACKGROUND: The incidence of gastric cancer in the Northern city, Hanoi is higher than in the Southern city, Ho Chi Minh, Vietnam. We previously reported that Helicobacter pylori vacA m1 genotype might be responsible for the difference between the two cities, however, the study only included non-cancer patients. The aim of this study is to investigate the non-cardia gastric cancer characteristics and the role of H. pylori virulence on different non-cardia gastric cancer incidence between two cities in Vietnam. METHODS AND RESULTS: We recruited 282 non-cardia gastric cancer patients that had undergone gastroscopy in two cities, Ho Chi Minh and Hanoi, Vietnam. Characteristics of non-cardia gastric cancer were late age of onset (mean age, 62.5 years), predominance in males (ratio of males/females; 3.9:1), diffuse type (55.3%), and high prevalence of H. pylori infection (79.4%). H. pylori infection and the vacA m1 genotype conferred an increased risk for GC (OR, 2.02; 95% CI 1.4-3.0; P = 0.0003 and OR, 2.7; 95% CI 1.5-4.7; P = 0.001, respectively). Interestingly, the presence of vacA m1 genotype in the gastric cancer group was significantly higher than that in the non-cancer group (68.8% vs 44.9%, P = 0.001) and the significant tendency still observed in Ho Chi Minh (67.6% vs 31.9%, P < 0.0001). CONCLUSION: We first describe the characteristics of non-cardia gastric cancer in Vietnam. Helicobacter pylori infection was associated with the development of non-cardia GC. vacA m1 genotype might contribute to incidence differences between the two cities.