Women with chronic follicular gastritis positive for Helicobacter pylori express lower levels of GKN1.

In women, serum levels of CTSB, GKN2, LIPF, LIPFG, AZGP1, TOP2A and PGA4 are proposed as predictive markers of gastric cancer. It is unknown whether GKN1 expression varies with the sex of patients with chronic gastritis or gastric cancer. We studied 36 patients with histopathological diagnosis of chronic gastritis from the state of Guerrero, Mexico. PCR was performed for H. pylori detection and GKN1 expression was determined by RT-qPCR and western blot. GKN1 mRNA expression was significantly lower in patients with chronic follicular gastritis than in those with chronic chemical gastritis (p = 0.00071). The mRNA and protein level of expression of GKN1 were significantly lower in women with chronic follicular gastritis than in men with the same condition (p = 0.0279 and p = 0.0014, respectively); the lowest levels of GKN1 were detected in women with H. pylori-positive follicular gastritis (p = 0.0175 and p = 0.0111, respectively). Through a bioinformatic analysis, estrogen response elements were identified in the GKN1 promoter.

Helicobacter pylori inhibits GKN1 expression via the CagA/p-ERK/AUF1 pathway.

BACKGROUND: Recent studies have shown that gastrokine 1 (GKN1), an important tumor suppressor gene, is downregulated in Helicobacter pylori (H. pylori) infected gastric mucosa and gastric cancer. However, the underlying mechanism is poorly understood. Herein, we investigated the potential mechanism of H. pylori-induced GKN1 downregulation. MATERIALS AND METHODS: GKN1 and AU-rich element RNA-binding factor 1 (AUF1) expressions were assessed by quantitative real-time PCR, Western blot, or immunohistochemistry in H. pylori-infected tissues and H. pylori co-cultured cell lines. The regulation of AUF1 on GKN1 was determined by RNA pulldown assay, RNA immunoprecipitation, mRNA turnover, and luciferase activity assays. The involvement of phosphorylated extra-cellular signal-regulated kinase (p-ERK) or CagA in H. pylori-induced AUF1 expression was verified using p-ERK inhibitor or CagA knockout H. pylori. In addition, the cell proliferation and migration capacities of AUF1-knockdown cells were investigated. RESULTS: GKN1 expression progressively decreased from H. pylori-infected gastritis to gastric cancer tissues. H. pylori co-culture also induced significant GKN1 reduction in GES-1 and BGC-823 cells. Besides, the mRNA level of GKN1 and AUF1 in human gastric mucosa showed negative correlation significantly. AUF1 knockdown resulted in upregulation of GKN1 expression and promoted GKN1 mRNA decay by binding the 3' untranslated region of GKN1 mRNA H. pylori-induced AUF1 expression was associated with p-ERK activation and CagA. Furthermore, knockdown of AUF1 significantly inhibited cell viability, migration ability, and arrested fewer cells in S-phase. CONCLUSION: Our data demonstrated that H. pylori infection downregulated GKN1 expression via the CagA/p-ERK/AUF1 pathway. AUF1 promoted gastric cancer at least partly through downregulating GKN1, which presented a novel potential target for the treatment of gastric cancer.

GKN1 expression in gastric cancer cells is negatively regulated by miR-544a.

Gastrokine1 (GKN1), important for maintaining the physiological function of the gastric mucosa, is highly expressed in the stomach of healthy individuals but is down-regulated or absent in gastric tumor tissues and derived cell lines. The mechanisms underlying GKN1 gene inactivation are still unknown. We previously showed that GKN1 downregulation in gastric tumors is likely associated with an epigenetic transcriptional complex that negatively regulates GKN1 expression. In addition, TSA-mediated inhibition of HDACs leads to GKN1 restoration at the transcriptional level, but no at the translational level. These findings led to hypothesize the activation of a second regulatory mechanism microRNAs-mediated, thus resulting in translational repression and gene silencing. Bioinformatic analyses performed with 5 different algorithms highlighted that 4 miRNAs contained a seed sequence for the 3'UTR of GKN1 mRNA. Among these, only two miRNAs, hsa-miR-544a and miR-1245b-3p directly target the GKN1-3'UTR as evaluated by luciferase reporter assays. TaqMan miRNA assay performed on gastric cancer cell lines after TSA treatment showed a stronger increase of miR-544a expression than that of miR-1245b-3p. Finally, co-transfection of AGS cells with GKN1-3'UTR and premiR-544a showed compared to controls, a strong reduction of GKN1 expression both at translational and transcriptional levels. The up-regulation of miR-544a could be crucially involved in the GKN1 translational repression, thus suggesting its potential role as a biomarker and therapeutic target in GC patients. These findings indicate that epigenetic mechanisms leading to the inactivation of GKN1 play a key role in the multi-step process of gastric carcinogenesis and would provide an essential starting point for the development of new therapeutic strategies based on epigenetic targets for alternatives gene.

The role and diagnostic potential of gastrokine 1 in gastric cancer.

INTRODUCTION: Gene for gastrokine 1 (GKN1) was identified as one of the most significant in gastric cancer and indicated as a potential therapeutic target. AIM: The aim was a review of literature reports concerning the role and diagnostic potential of GKN1 in gastric cancer. MATERIALS AND METHODS: PubMED database was searched for sources using the following keywords: gastrokine 1/GKN1/AMP-18 and gastric cancer, Helicobacter pylori, aspirin, nonsteroidal anti-inflammatory drugs. Preference was given to the sources which were published within the past 10 years. CONCLUSION: GKN1 is a stomach-specific protein, and its role consists of maintaining mucosal integrity as well as the replenishment of the surface lumen epithelial cells layer. The evaluation of GKN1 expression seems to be a useful indicator of the presence of neoplastic or inflammatory lesions in the gastric mucosa. GKN1 expression is decreased in gastric tumor tissues and derived cell lines and its upregulation in cell lines of gastric cancer induces cells apoptosis. The mechanism by which GKN1 is inactivated in gastric cancer cells is still not fully understood. The future diagnostic capabilities of gastric cancer concern the assessment of serum GKN1 concentration by means of ELISA method. Serum GKN1 concentration is not related to patients' sex. Moreover, the measurement of GKN1 concentration is possible only after the incubation of samples at 70°C for 10 minutes. Nevertheless, the aspect of quantitative serum GKN1 evaluation is new in the context of available literature and requires further studies.

NKX6.3 protects against gastric mucosal atrophy by downregulating ß-amyloid production.

BACKGROUND: Atrophic gastritis is characterized by loss of appropriate glands and reduction in gastric secretory function due to chronic inflammatory processes in gastric mucosa. Moreover, atrophic gastritis is considered as a precancerous condition of gastric cancer. However, little is known about the molecular mechanism underlying gastric mucosal atrophy and its contribution to gastric carcinogenesis. Thus, we hypothesized that transcription factor NKX6.3 might be involved in maintaining gastric epithelial homeostasis by regulating amyloid ß (Aß) production. AIM: To determine whether NKX6.3 might protect against gastric mucosal atrophy by regulating Aß production. METHODS: We identified NKX6.3 depletion induced cell death by cell count and Western blot assay. Production and mechanism of Aß oligomer were analyzed by enzyme-linked immunosorbent assay, Western blot, immunoprecipitation, real-time quantitative polymerase chain reaction and immunofluorescence analysis. We further validated the correlation between expression of NKX6.3, Helicobacter pylori CagA, Aß oligomer, apolipoprotein E (ApoE), and ß-secretase 1 (Bace1) in 55 gastric mucosae. RESULTS: NKX6.3 depletion increased both adherent and floating cell populations in HFE-145 cells. Expression levels of cleaved caspase-3, -9, and poly ADP ribose polymerase were elevated in floating HFE-145shNKX6.3 cells. NKX6.3 depletion produced Aß peptide oligomers, and increased expression of ApoE, amyloid precursor protein, Aß, Bace1, low-density lipoprotein receptor, nicastrin, high mobility group box1, and receptor for advanced glycosylation end product proteins. In immunoprecipitation assay, γ-secretase complex was stably formed only in HFE-145shNKX6.3 cells. In gastric mucosae with atrophy, expression of Aß peptide oligomer, ApoE, and Bace1 was detected and inversely correlated with NKX6.3 expression. Treatment with recombinant Aß 1-42 produced Aß oligomeric forms and decreased cell viability in HFE-145shNKX6.3 cells. Additionally, NKX6.3 depletion increased expression of inflammatory cytokines and cyclooxygenase-2. CONCLUSION: NKX6.3 inhibits gastric mucosal atrophy by regulating Aß accumulation and inflammatory reaction in gastric epithelial cells.

Evaluation of GKN1 and GKN2 gene expression as a biomarker of gastric cancer.

AIM: The aim of this study was to investigate the expression of GKN1 and GKN2 genes as probable biomarkers for gastric cancer. BACKGROUND: Gastric cancer is a multifactorial process characterized by the uncontrolled growth and dissemination of abnormal cells. Survival rates of gastric cancer tend to be poor, a plausible explanation is a combination of a late-stage diagnosis and limited access to treatment. In this regard, finding relevant and measurable biomarkers is urgently needed. METHODS: 27 samples of gastric cancer tissues were enrolled into this study, according to their pathological responses. The alteration of genes expression were evaluated by Real-Time PCR technique. RESULTS: Our findings showed the significant reduction of Gastrokin-1 and Gastrokine-2 genes expression in the cancerous specimens in comparison with the normal tissues. (P = 0.008 and P = 0.004 respectively). CONCLUSION: Our findings showed the significant reduction of Gastrokin-1 and Gastrokine-2 genes expression in the cancerous specimens in comparison with the normal tissues. (P = 0.008 and P = 0.004 respectively).

Role of human GKN1 on APP processing in gastric cancer.

Gastrokine 1 (GKN1) is highly expressed in gastric tissue and is secreted into the stomach but is not expressed in gastric cancer. GKN1 belongs to the BRICHOS domain family and plays a major role in maintaining gastric mucosa integrity. We previously demonstrated that a recombinant human GKN1 protein was able to interact with the amyloid precursor protein (APP) and was endowed with an anti-amyloidogenic property because it inhibited polymerization of the Aß(1-40) peptide released from APP upon its partial hydrolysis. Here, we report that GKN1 can act as a physiological suppressor of Aß production in gastric cancer cells. GKN1 blocked the access of γ-secretase to APP, thereby facilitating the cleavage of APP by α- and ß-secretases. GKN1 directly interacted with APP C-terminal fragments, C83 and C99. In addition, it did not affect γ-secretase activity in gastric cancer cells because it did not alter Notch1 processing. GKN1-mediated inhibition of APP processing might represent a new approach for the prevention and therapy of Alzheimer's disease (AD).

Epigenetic alterations of gastrokine 1 gene expression in gastric cancer.

The gastrokine 1 (GKN1) protein is important for maintaining the physiological function of the gastric mucosa. GKN1 is down-regulated in gastric tumor tissues and derived cell lines and its over-expression in gastric cancer cells induces apoptosis, suggesting a possible role for the protein as a tumor suppressor. However, the mechanism by which GKN1 is inactivated in gastric cancer remains unknown. Here, we investigated the causes of GKN1 silencing to determine if epigenetic mechanisms such as histonic modification could contribute to its down-regulation. To this end, chromatin immunoprecipitation assays for the trimethylation of histone 3 at lysine 9 (H3K9triMe) and its specific histone-lysine N-methyltransferase (SUV39H1) were performed on biopsies of normal and cancerous human gastric tissues. GKN1 down-regulation in gastric cancer tissues was shown to be associated with high levels of H3K9triMe and with the recruitment of SUV39H1 to the GKN1 promoter, suggesting the presence of an epigenetic transcriptional complex that negatively regulates GKN1 expression in gastric tumors. The inhibition of histone deacetylases with trichostatin A was also shown to increase GKN1 mRNA levels. Collectively, our results indicate that complex epigenetic machinery regulates GKN1 expression at the transcriptional level, and likely at the translational level.

Gastrokine 1 inhibits gastric cancer cell migration and invasion by downregulating RhoA expression.

BACKGROUND: We investigated whether GKN1, a gastric tumor suppressor, contributes to the progression of gastric cancer by regulating RhoA expression. METHODS: We analyzed the expression of GKN1, RhoA, miR-185, and miR-34a in 35 gastric cancer tissues, and compared their expression with T category and TNM stage. Cell migration and invasion, as well as the expression of epithelial-to-mesenchymal transition (EMT)-related proteins, were assessed in GKN1- and RhoA small interfering RNA (siRhoA)-transfected and recombinant-GKN1-treated AGS and MKN1 gastric cancer cells. RESULTS: Expression of RhoA protein and messenger RNA (mRNA) was increased in 15 (42.9 %) and 17 (48.6 %) of 35 gastric cancer tissues respectively, and was associated with higher T category and TNM stage. GKN1 expression was significantly decreased in 27 gastric cancers (77.1 %) with a higher T category, and was inversely correlated with RhoA mRNA expression. In AGS and MKN1 cells, GKN1 expression increased miR-185 and miR-34a expression and reduced RhoA mRNA and protein expression. A positive relationship between GKN1 and miR-34a and miR-185 expression and an inverse relationship between miR-34a and RhoA expression were observed in gastric cancer tissues. Cell migration and invasiveness were markedly decreased in GKN1- and siRhoA-transfected cells. GKN1 expression and silencing of RhoA decreased the expression of the proteins Snail, Slug, and vimentin. Furthermore, miR-185 and miR-34a silencing in MKN1 cells transfected with GKN1 stimulated cell migration and invasion, and increased the expression of EMT-related proteins. CONCLUSION: Our data suggest that GKN1 may inhibit gastric cancer cell migration and invasion by downregulating RhoA expression in a miR-185- and miR-34a-dependent manner.

Gastrokine 1 mRNA in human sera is not informative biomarker for gastric cancer.

BACKGROUND: We aimed to ascertain if Gastrokine 1 mRNA in the sera of patients with gastric cancer might be an informative biomarker for the disease. RESULTS: Analysis of GKN1 mRNA in serum samples from healthy individuals (n = 23) and from patients with diagnosis of gastric cancer (n = 16), performed by using absolute quantification based on standard curve method, did not show any significative statistical difference between the two unpaired group of individuals. CONCLUSIONS: Our preliminary results did not confirm GKN1 as a potential biomarker for gastric cancer.

Ectopic expression of gastrokine 1 in gastric cancer cells up-regulates tight and adherens junction proteins network.

Gastrokine 1 (GKN1) is a stomach-specific protein important in the replenishment of the surface lumen epithelial cell layer and in maintaining mucosal integrity. A role in cell proliferation and differentiation has also been hypothesized. Despite these findings, the function(s) as well as the cellular localization of GKN1 in the cellular machinery are currently not clarified. The investigation of subcellular localization of GKN1 in gastric cancer cells can provide insights into its potential cellular roles. Subcellular fractions of gastric cancer cells (AGS) transfected with full-length GKN1 (flGKN1) or incubated with recombinant GKN1 (rGKN1) lacking the first 20 amino acids at N-terminal were analyzed by Western blot and confocal microscopy and compared with those from normal gastric tissue. Wild type GKN1 (wtGKN1) and flGKN1 were revealed in the cytoplasm and in the membrane fractions of gastric cells, whereas rGKN1 was revealed in the cytoplasmic fractions, but a high amount was detected in the membrane pellet of the AGS lysate. The cellular distribution of GKN1 was also confirmed by confocal microscopy. The purified protein was also used to highlight its possible association with actin through confocal microscopy, pelleting assay, and size-exclusion chromatography. GKN1 co-localizes with actin in normal gastric tissue, but no direct interaction was observed between the two proteins in vitro. Most likely, GKN1 indirectly participates in actin stabilization since its overexpression in gastric cancer cells strongly increases the expression of tight and adherens junction proteins.

Decreased expression of gastrokine 1 in gastric mucosa of gastric cancer patients.

AIM: To investigate the expression of gastrokine 1 (GKN1) in normal gastric mucosa, precancerous lesions and gastric cancer tissues, and to analyse its correlations with tumour site and pathological pattern. METHODS: Thirty gastric cancer patients (12 cases of diffuse type and 18 cases of intestinal type), 13 atrophic gastritis patients and 15 healthy volunteers with almost normal gastric mucosa (superficial gastritis) were enrolled in this study. Helicobacter pylori (H. pylori) infection was examined in all subjects. All gastric mucosa biopsy specimens were obtained. Cancer-adjacent specimens were taken from corresponding gastric cancer patients. Immunohistochemistry and real-time PCR were performed to determine the expressions of the GKN1 protein and mRNA, respectively. RESULTS: H. pylori infection had no significant association with age, gender, tumour site or pathological pattern in all subjects. Compared with the superficial gastritis and atrophic gastritis groups, the expression of GKN1 protein (P = 0.011) and mRNA (P < 0.001) in gastric cancer was significantly decreased. The GKN1 mRNA level in diffuse type gastric cancer was significantly lower than in intestinal type gastric cancer (0.296 ± 0.076 vs 0.525 ± 0.164, P < 0.001). CONCLUSION: Compared with almost normal gastric mucosa, GKN1 expression in the gastric mucosa of gastric cancer patients is decreased; this is associated with progression and prognosis of gastric cancer.

Anti-amyloidogenic property of human gastrokine 1.

Gastrokine 1 (GKN1) is a stomach-specific protein expressed in normal gastric tissue but absent in gastric cancer. GKN1 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids also found in several unrelated proteins associated with major human diseases like BRI2, related to familial British and Danish dementia and surfactant protein C (SP-C), associated with respiratory distress syndrome. It was reported that recombinant BRICHOS domains from BRI2 and SP-C precursor (proSP-C) prevent fibrils formation of amyloid-beta peptide (Aß), that is the major component of extracellular amyloid deposits in Alzheimer's disease. Here we investigated on the interaction between human recombinant GKN1 (rGKN1) and Aß peptide (1-40) that derives from the partial hydrolysis of the amyloid precursor protein (APP). GKN1 prevented amyloid aggregation and fibrils formation by inhibiting Aß(1-40) polymerization, as evaluated by SDS-PAGE, thioflavin-T binding assay and gel filtration experiments. Mass spectrometry showed the formation of a prevailing 1:1 complex between GKN1 and Aß(1-40). SPR analysis of GKN1/Aß interaction led to calculate a dissociation constant (KD) of 34 µM. Besides its interaction with Aß(1-40), GKN1 showed also to interact with APP as evaluated by confocal microscopy and Ni-NTA pull-down. Data strongly suggest that GKN1 has anti-amyloidogenic properties thus functioning as a chaperone directed against unfolded segments and with the ability to recognize amyloidogenic polypeptides and prevent their aggregation.

The tumor suppressor gastrokine-1 is expressed in placenta and contributes to the regulation of trophoblast migration.

INTRODUCTION: Gastrokine-1 (GKN1) is a secreted auto-/paracrine protein, described to be expressed in the gastric mucosa. In gastric cancers GKN1 expression is commonly down-regulated. While current research focusses on the exploration of tumor-suppressive properties of GKN1 with regard to its potential clinical use in the treatment of gastroenterologic tumor disease, nothing is known about GKN1 expression and function in other organ systems. We investigated GKN1 expression in placental tissue and cells. MATERIALS AND METHODS: GKN1 was localized using immunohistochemistry in first and third trimester placental tissue, hydatidiform moles and various gestational trophoblastic neoplasias. We determined the expression of GKN1 in immunomagnetic bead-separated term placental cells and in choriocarcinoma cell lines. The role of GKN1 for JEG-3 migration was studied using live cell imaging. E-cadherin, MMP-2 and -9, TIMP-1 and -2, as well as urokinase (uPA) expression levels were determined. RESULTS: GKN1 is expressed in healthy third trimester placentas. Its expression is specifically limited to the extravillous trophoblast (EVT). GKN1 expression is significantly reduced in choriocarcinoma cell lines and gestational trophoblastic neoplasias. GKN1 attenuates the migration of JEG-3 choriocarcinoma cells in vitro, possibly via AKT-mediated induction of E-cadherin. GKN1 treatment reduced MMP-9 expression in JEG-3. DISCUSSION: Besides its role in gastric physiology our results clearly indicate regulatory functions of GKN1 in the EVT at the feto-maternal interface during pregnancy. Based on our findings in the JEG-3 choriocarcinoma cell line, an auto-/paracrine role of GKN1 for EVT motility and villous anchorage at the basal plate is conceivable. Thus, the tumor suppressor GKN1 is expressed in placental EVT and might contribute to the regulation of EVT migration/invasion.

Gastrokine 1 expression in the human gastric mucosa is closely associated with the degree of gastritis and DNA methylation.

PURPOSE: Gastrokine 1 plays an important role in gastric mucosal defense. Additionally, the Gastrokine 1-miR-185-DNMT1 axis has been shown to suppress gastric carcinogenesis through regulation of epigenetic alteration. Here, we investigated the effects of Gastrokine 1 on DNA methylation and gastritis. MATERIALS AND METHODS: Expression of Gastrokine 1, DNMT1, EZH2, and c-Myc proteins, and the presence of Helicobacter pylori CagA protein were determined in 55 non-neoplastic gastric mucosal tissue samples by western blot analysis. The CpG island methylation phenotype was also examined using six markers (p16, hMLH1, CDH1, MINT1, MINT2 and MINT31) by methylation-specific polymerase chain reaction. Histological gastritis was assessed according to the updated Sydney classification system. RESULTS: Reduced Gastrokine 1 expression was found in 20 of the 55 (36.4%) gastric mucosal tissue samples and was closely associated with miR-185 expression. The Gastrokine 1 expression level was inversely correlated with that of DNMT1, EZH2, and c-Myc, and closely associated with the degree of gastritis. The H. pylori CagA protein was detected in 26 of the 55 (47.3%) gastric mucosal tissues and was positively associated with the expression of DNMT1, EZH2, and c-Myc. In addition, 30 (54.5%) and 23 (41.9%) of the gastric mucosal tissues could be classified as CpG island methylation phenotype-low and CpG island methylation phenotype-high, respectively. Reduced expression of Gastrokine 1 and miR-185, and increased expression of DNMT1, EZH2, and c-Myc were detected in the CpG island methylation phenotype-high gastric mucosa. CONCLUSIONS: Gastrokine 1 has a crucial role in gastric inflammation and DNA methylation in gastric mucosa.

Gastrokine 1 Expression in the Human Gastric Mucosa Is Closely Associated with the Degree of Gastritis and DNA Methylation

PURPOSE: Gastrokine 1 plays an important role in gastric mucosal defense. Additionally, the Gastrokine 1-miR-185-DNMT1 axis has been shown to suppress gastric carcinogenesis through regulation of epigenetic alteration. Here, we investigated the effects of Gastrokine 1 on DNA methylation and gastritis. MATERIALS AND METHODS: Expression of Gastrokine 1, DNMT1, EZH2, and c-Myc proteins, and the presence of Helicobacter pylori CagA protein were determined in 55 non-neoplastic gastric mucosal tissue samples by western blot analysis. The CpG island methylation phenotype was also examined using six markers (p16, hMLH1, CDH1, MINT1, MINT2 and MINT31) by methylation-specific polymerase chain reaction. Histological gastritis was assessed according to the updated Sydney classification system. RESULTS: Reduced Gastrokine 1 expression was found in 20 of the 55 (36.4%) gastric mucosal tissue samples and was closely associated with miR-185 expression. The Gastrokine 1 expression level was inversely correlated with that of DNMT1, EZH2, and c-Myc, and closely associated with the degree of gastritis. The H. pylori CagA protein was detected in 26 of the 55 (47.3%) gastric mucosal tissues and was positively associated with the expression of DNMT1, EZH2, and c-Myc. In addition, 30 (54.5%) and 23 (41.9%) of the gastric mucosal tissues could be classified as CpG island methylation phenotype-low and CpG island methylation phenotype-high, respectively. Reduced expression of Gastrokine 1 and miR-185, and increased expression of DNMT1, EZH2, and c-Myc were detected in the CpG island methylation phenotype-high gastric mucosa. CONCLUSIONS: Gastrokine 1 has a crucial role in gastric inflammation and DNA methylation in gastric mucosa.