Normal Gastric Epithelial Cell Line Research Articles

Transient receptor potential vanilloid 4 (TRPV4) silencing in Helicobacter pylori‐infected human gastric epithelium

Background Helicobacter pylori (HP) infection induces methylation silencing of specific genes in gastric epithelium. Various stimuli activate the nonselective cation channel TRPV4, which is expressed in gastric epithelium where it detects mechanical stimuli and promotes ATP release. As CpG islands in TRPV4 are methylated in HP‐infected gastric epithelium, we evaluated HP infection‐dependent changes in TRPV4 expression in gastric epithelium.Materials and MethodsHuman gastric biopsy samples, a human gastric cancer cell line (AGS), and a normal gastric epithelial cell line (GES‐1) were used to detect TRPV4 mRNA and protein expression by RT‐PCR and Western blotting, respectively. Ca2+ imaging was used to evaluate TRPV4 ion channel activity. TRPV4 methylation status was assessed by methylation‐specific PCR (MSP). ATP release was measured by a luciferin‐luciferase assay.Results TRPV4 mRNA and protein were detected in human gastric biopsy samples and in GES‐1 cells. MSP and demethylation assays showed TRPV4 methylation silencing in AGS cells. HP coculture directly induced methylation silencing of TRPV4 in GES‐1 cells. In human samples, HP infection was associated with TRPV4 methylation silencing that recovered after HP eradication in a time‐dependent manner.Conclusion HP infection‐dependent DNA methylation suppressed TRPV4 expression in human gastric epithelia, suggesting that TRPV4 methylation may be involved in HP‐associated dyspepsia.

Collapsin response mediator protein 4 isoforms (CRMP4a and CRMP4b) have opposite effects on cell proliferation, migration, and invasion in gastric cancer.

BackgroundCollapsin response mediator proteins (CRMPs) were originally identified in the nervous system and are involved in neuronal development. Similar to CRMP1, CRMP4 has a shorter transcript encoding a short isoform known as CRMP4a, and a longer transcript encoding a long isoform known as CRMP4b. Previous studies have shown that CRMP4a and CRMP4b exhibit opposing functions in neurite outgrowth. In the present study, we aimed to determine whether CRMP4a and CRMP4b have divergent effects in gastric cancer.MethodsWe first analyzed the mRNA and protein expression levels of CRMP4a and CRMP4b in surgical resected specimens, gastric cancer cell lines and normal gastric epithelial cell line GES-1 by quantitative real-time PCR. Open reading frame and CRMP4b shRNA were generated by lentivirus package and stable cells stably expressing CRMP4a open reading frame and CRMP4b shRNA were constructed. Then the roles of CRMP4a and CRMP4b in cell proliferation, cell cycle progression, apoptosis, migration, invasion, and adhesion were determined by cell proliferation assays, flow cytometry analysis, transwell migration and invasion assays, cell Adhesion Assay, and tumorigenicity assays in nude mice, respectively.ResultsCRMP4a expression was lower and CRMP4b expression was higher in tumor tissue samples as compared to paired non-tumor tissue samples. Additionally, CRMP4a expression was lower and CRMP4b expression was higher in gastric cancer cell lines than in the normal gastric epithelial cell line GES-1. CRMP4a overexpression and CRMP4b silencing suppressed cell proliferation in vitro and in vivo. Additionally, CRMP4a overexpression and CRMP4b silencing induced a significant G1-phase arrest and a decrease of the percentage of cells in S-phase. Furthermore, CRMP4a overexpression and CRMP4b silencing inhibited cell migration, invasion, and adhesion. However, neither CRMP4a overexpression nor CRMP4b silencing affected apoptosis.ConclusionThese results indicate that CRMP4a and CRMP4b have opposite effects on cell proliferation, migration, and invasion in gastric cancer.

Electrochemical biosensor for protein kinase A activity assay based on gold nanoparticles-carbon nanospheres, phos-tag-biotin and β-galactosidase

A sensitive and selective electrochemical biosensor was fabricated for protein kinase A (PKA) activity assay. Multiple signal amplification techniques were employed including the nanocomposite of gold nanoparticles and carbon nanospheres (Au@C), the biocomposite of SiO2 and streptavidin (SiO2-SA), the composite of AuNPs and biotinylated β-galactosidase (AuNPs-B-Gal) and in situ enzymatic generation of electrochemical activity molecule of p-aminophenol. After peptides were assembled on Au@C modified electrode surface, they were phosphorylated by PKA in the presence of ATP. Then, biotinylated Phos-tag was modified on electrode surface through the specific interaction between Phos-tag and phosphate group. Finally, SiO2-SA and AuNPs-B-Gal were captured through the specific interaction between biotin and streptavidin. Because the electrochemical response of p-aminophenol was directly related to PKA concentration, an innovative electrochemical assay could be realized for PKA detection. The detection limit was 0.014unit/mL. The developed method showed high detection sensitivity and selectivity. In addition, the fabricated biosensor can be also applied to detect PKA in human normal gastricepithelial cell line and human gastric carcinoma cell line with satisfactory results.

Overexpressed MALAT1 promotes invasion and metastasis of gastric cancer cells via increasing EGFL7 expression

ObjectiveLong non-coding RNAs (lncRNAs) have been demonstrated to participate in various cancers. Here, the role and its potential mechanism of MALAT1 in invasion and migration of gastric cancer (GC) were investigated. MethodsGastric adenocarcinoma tissues and matched normal adjacent tissues were isolated from 25 patients with GC. The expression of epidermal growth factor-like domain-containing protein 7 (EGFL7) was detected in the normal gastric mucosa epithelial GES-1 cell line and three different differentiation GC cell lines, including MKN28 (well-differentiated adenocarcinoma), SGC7901 (moderately differentiated adenocarcinoma) and BGC823 (poorly differentiated adenocarcinoma). Tumor xenotransplant mouse model was established with the injection of cell line pretreated with lentiviral vectors for si-MALAT1 or si-control. ResultsThe expression of MALAT1 was up-regulated in GC tissues and three cell lines. Si-MALAT1/pcDNA-MALAT1 induced the decrease of cell invasion and migration, while the effects were reversed by the transfection of pcDNA-EGFL7/si-EGFL7. ChIP assay showed that MALAT1 regulated EGFL7 expression by altering the level of H3 histone acetylation in EGFL7 promoter. In tumor xenotransplant mice, down-regulated MALAT1 contributed to the inhibition of tumor metastasis. ConclusionsUp-regulated MALAT1 promoted the invasion and metastasis of GC, and the increase of EGFL7 expression was a potential mechanism via altering its H3 histone acetylation level.

Comparative Proteomic Profiling of Extracellular Proteins between Normal and Gastric Cancer Cells.

Gastric cancer is the second leading cause of cancer-related deaths worldwide. Gastric cancer is often detected at a late stage when treatment is difficult. Biomarkers for early detection and drug targets for gastric cancer therapy are critical for effective management of gastric cancer. Secreted proteins not only play integral roles in cancer progression and metastasis, they are also easily accessible. Secreted proteins within the tumor microenvironment are therefore an attractive source of biomarkers and drug targets. In this study, iTRAQ-based liquid chromatography/tandem mass spectrometry was used for comparative profiling of the secretomes of 11 gastric cancer cell lines versus a normal gastric epithelial cell line. Of the close to 800 proteins detected, about 600 proteins were detected to display differential expression in one or more gastric cancer cell lines compared to normal cells. These differentially expressed proteins predominantly have binding or enzymatic activities and are largely associated with cellular and metabolic processes. Overexpression of ARPC4 was validated in gastric cell lines and its novel function in gastric cancer cell migration and invasion demonstrated in vitro. The findings support the notion of ARPC4 as a potential biomarker/drug target for metastatic gastric cancer.

The natural secolignan peperomin E induces apoptosis of human gastric carcinoma cells via the mitochondrial and PI3K/Akt signaling pathways in vitro and in vivo

BackgroundPeperomin E (PepE) is a type of secolignan that is a major component of the plant Peperomia dindygulensis. It has been shown to exert anticancer effects in various cancer cell lines; however, the effects of PepE on human gastric cancer remain unexplored. PurposeThe aim of this study was to investigate the effectiveness of PepE as a treatment of gastric cancer and to identify the underlying mechanisms of its anticancer activity. Study designThe efficacy of PepE was examined using human gastric carcinoma SGC-7901, BGC-823, MKN-45 cell lines and normal gastric epithelial GES-1 cell line as an in vitro model and SGC-7901 xenograft mice as an in vivo model. MethodsCell viability assays were used to examine the anticancer effect of 0–204.8µM concentrations of PepE in vitro. Additionally, flow cytometry and western blotting were used to elucidate the mechanism with a particular focus on apoptosis. SGC-7901 cells were injected into BALB/c mice, which were then treated with 5 or 15mg/kg/day dose of PepE. The in vivo activity of PepE was investigated by measuring tumors and conducting immunohistochemistry experiments. The safety of PepE was investigated by measuring blood biochemical parameters and conducting histopathological analysis. Taxol was used throughout as a positive control. ResultsThe results showed that PepE exhibited antiproliferative effects against gastric cancer cells and induced their apoptosis in a dose dependent manner with lower toxicity against normal gastric epithelial cells. Mechanistic evaluations indicated that PepE induced apoptosis by reducing the mitochondrial membrane potential (MTP), inducing cytochrome C release from mitochondria, reducing the ratio of Bcl-2/Bax and Bcl-xl/Bad, increasing activation of caspase-3, and decreasing the levels of PI3K and pAkt. The apoptotic effect of PepE on SGC-7901 cells was partially blocked by an Akt activator SC79. PepE potently inhibited in vivo tumor growth with no obvious toxicity following subcutaneous inoculation of SGC-7901 cells in nude mice. ConclusionsThese findings indicate that PepE can inhibit cell proliferation and induce apoptosis of gastric cancer cells through mitochondrial and PI3K/Akt signaling pathways with relative safety and may be a novel effective chemotherapeutic agent against gastric cancer.

Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells.

BackgroundThe transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells.MethodsThe expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay.ResultsHigh levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation and invasion, arrested the cell cycle, and induced apoptosis in vitro.ConclusionThe asymmetric mono-carbonyl analog of curcumin Da0324 exhibited significantly improved antigastric cancer activity. Da0324 may be a promising NF-κB inhibitor for the selective targeting of cancer cells. However, further studies are needed in animals to validate these findings for the therapeutic use of Da0324.

Long non-coding antisense RNA KRT7-AS is activated in gastric cancers and supports cancer cell progression by increasing KRT7 expression.

Alterations in long non-coding RNAs (lncRNAs) are associated with human carcinogenesis. One group of lncRNAs, which are antisense in orientation to coding mRNAs (ASs), have been recently described in cancers but are poorly understood. We sought to identify ASs involved in human gastric cancer (GC) and to elucidate their mechanisms of action in carcinogenesis. We performed massively parallel RNA sequencing in GCs and matched normal tissues, as well as in GC-derived and normal gastric epithelial cell lines. One AS, designated KRT7-AS, was selected due to its marked upregulation and concordant expression with its cognate sense counterpart, KRT7, in GC tissues and cell lines. KRT7-AS formed an RNA-RNA hybrid with KRT7 and controlled KRT7 expression at both the mRNA and the post-transcriptional levels. Moreover, forced overexpression of the KRT7-overlapping region (OL) of KRT7-AS (but not its non-KRT7-overlapping portions) increased keratin 7 protein levels in cells. Finally, forced overexpression of full-length (FL) KRT7-AS or OL KRT7-AS (but not its non-KRT7-overlapping regions) promoted GC cell proliferation and migration. We conclude that lncRNA KRT7-AS promotes GC, at least in part, by increasing KRT7 expression.

Transient receptor potential vanilloid 4-dependent calcium influx and ATP release in mouse and rat gastric epithelia.

To explore the expression of transient receptor potential vanilloid 4 (TRPV4) and its physiological meaning in mouse and rat gastric epithelia. RT-PCR and immunochemistry were used to detect TRPV4 mRNA and protein expression in mouse stomach and a rat normal gastric epithelial cell line (RGE1-01), while Ca(2+)-imaging and electrophysiology were used to evaluate TRPV4 channel activity. ATP release was measured by a luciferin-luciferase assay. Gastric emptying was also compared between WT and TRPV4 knockout mice. TRPV4 mRNA and protein were detected in mouse tissues and RGE1-01 cells. A TRPV4-specific agonist (GSK1016790A) increased intracellular Ca(2+) concentrations and/or evoked TRPV4-like current activities in WT mouse gastric epithelial cells and RGE1-01 cells, but not TRPV4KO cells. GSK1016790A or mechanical stimuli induced ATP release from RGE1-01 cells while TRPV4 knockout mice displayed delayed gastric emptying in vivo. TRPV4 is expressed in mouse and rat gastric epithelium and contributes to ATP release and gastric emptying.

Inhibition of cell proliferation in gastric cancer cell lines on exposure to rubriflordilactone A

<p class="Abstract">The present study investigates the effect of rubriflordilactone A on the viability and its underlying mechanism in gastric cancer cell lines (SNU-1 and SNU-5) and normal gastric epithelial cell line (GES‑1). Incubation of the gastric cancer and non cancer cell lines in acidic media led to reduction in the viability of the non cancer cells without any effect on cancer cells. Apoptosis in SNU-1 and SNU-5 cells was induced on exposure to rubriflordilactone A after 48 hours compared to the control cells (p<0.01). The percentage of apoptosis in SNU-1 and SNU-5 cells on exposure to rubriflordilactone A was 79.3 ± 4.7 and 74.0 ± 5.1, respectively after 48 hours. Exposure of SNU-1 and SNU-5 cancer cell lines to rubriflordilactone A at a concentration of 10 μM in media with acidic pH decreased phosphorylation of ERK ½. The similar reduction was caused by ERK 1/2 phosphorylation inhibition, PD98059. Thus rubriflordilactone A reduces viability of gastric cancer cell lines by inducing apoptosis through the reduction of ERK 1/2 phosphorylation.</p><p> </p>

MicroRNA-101 induces apoptosis in cisplatin-resistant gastric cancer cells by targeting VEGF-C.

Deregulation of microRNAs (miRNAs) is known to be associated with drug resistance in human cancers. However, the precise role of miR‑101 in the cisplatin (DPP) resistance of human gastric cancer cells has not been elucidated, yet. The present study revealed that miR‑101 was markedly downregulated in gastric cancer cell lines compared to that in the normal gastric mucosa epithelial cell line GES1. Furthermore, a significant decrease in miR‑101 levels, accompanied with an increased expression of vascular endothelial growth factor (VEGF)‑C in DDP‑resistant SGC7901 gastric cancer cells (SGC7901/DDP) compared with those in native SGC7901 cells was observed. In addition, forced overexpression of miR‑101 significantly inhibited cell proliferation, while enhancing cisplatin‑induced apoptosis of SGC7901/DDP cells. A luciferase reporter assay confirmed that VEGF‑C was a direct target of miR‑101 in SGC7901/DDP cells. Forced overexpression of miR‑101 in SGC7901/DDP cells reduced the expression of VEGF‑C, while knockdown of miR‑101 expression significantly enhanced VEGF‑C expression in SGC7901/DDP cells. Finally, overexpression of VEGF‑C inhibited DDP‑induced apoptosis in SGC7901 cells. In conclusion, the results of the present study suggested that miR‑101 inhibited the proliferation and promoted DDP‑induced apoptosis of DDP‑resistant gastric cancer cells, at least in part via targeting VEGF-C.

A novel long noncoding RNA-LOWEG is low expressed in gastric cancer and acts as a tumor suppressor by inhibiting cell invasion.

Long noncoding RNA (lncRNA) have been reported to be involved in the development of multiple cancers. The aim of this study was to report the identification of lncRNA-CTD-2108O9.1, which we have named lncRNA low expressed in gastric cancer (lncRNA-LOWEG), and investigate its role in cancer development. Total RNA was extracted from the tissues of 94 patients with GC, one normal gastric epithelial cell line and four GC cell lines. Expression levels of lncRNA-LOWEG were determined by real-time PCR. Moreover, CCK-8 proliferation assay, transwell cell invasion assay and flow cytometry were performed to study the effects of lncRNA-LOWEG on SGC-7901 cell proliferation, cell invasion and cell cycle progression. Lastly, western blot and real-time PCR were used to verify the potential target genes of lncRNA-LOWEG. Significantly reduced expression of lncRNA-LOWEG was found in gastric cancer tissues and cell lines (SGC-7901, AGS, BGC-823 and HG-27) compared with patient-matched nontumorous adjacent tissues (P < 0.01) or the normal gastric cell line GES-1 (P < 0.05). Moreover, the transwell assay showed that the number of cells capable of passing through the Matrigel was significantly reduced after lncRNA-LOWEG transfection (P < 0.05). However, lncRNA-LOWEG overexpression did not significantly influence cell proliferation (P > 0.05) and cell cycle progression (P > 0.05). Lastly, western blot and real-time PCR analysis suggested that lncRNA-LOWEG is positively correlated with the expression of leukemia inhibitory factor receptor (LIFR) gene at the translational level. LncRNA-LOWEG is a tumor suppressor that inhibits GC cell invasion. And LIFR gene is up-regulated by lncRNA-LOWEG.

Expression of NEK-6 in gastric cancer and its clinical significance

To explore the NEK-6 expression in gastric cancer tissue and its relationship with clinicopathological features. Fluorescent quantification PCR and Western blotting were used to examine the NEK-6 expression in 36 samples of fresh gastric cancer tissues and para-cancer gastric mucosal tissues, human gastric cancer cell lines(BGC-823, MKN-28, SGC-7901, MGC-803, HGC-27, AGS), and human normal gastric epithelial cell line (GES-1). Gastric cancer cell lines with the highest expression level were selected to perform the invasion and migration tests, and the effect of down-regulated NEK-6 expression by siRNA transfection on above invasion and migration tests were observed. Meanwhile NEK-6 expression in 94 paraffin samples of gastric cancer tissues was examined by immunohistochemistry and its positivity was compared among different clinicopathologic features. Fluorescent quantification PCR revealed gastric cancer tissues had significantly higher NEK-6 expression than para-cancer tissues(0.002 80±0.001 36 vs. 0.001 91±0.001 48, P<0.05), NEK-6 expression was up-regulated in 31 gastric cancer tissues (86.1%), and human gastric cancer cell lines had significantly higher NEK-6 expression than GES-1 cells, among whom BGC-823 and AGS cell lines were the highest. Invasion and migration tests showed that as compared to negative siRNA control group, ability of invasion and migration in BGC-823 and AGS cells after siRNA transfection was obviously decreased. In 94 paraffin samples, positive expression rate of NEK-6 was 60.6%(57/94), and NEK-6 expression was significantly associated with gastric cancer distant metastasis, lymph nodes metastasis and TNM staging(all P<0.05). NEK-6 expression is up-regulated in gastric cancer tissues, which is significantly associated with distant metastasis, lymph nodes metastasis and TNM staging. Down-regulation of NEK-6 expression can inhibit the ability of invasion and migration in gastric cancer cells.

In vitro and in vivo study on the regulation of microRNA-101 in human gastric cancer proliferation, migration and invasion

Objective To investigate the expression of microRNA-101 (miRNA-101) in human gastric cancer, and to explore its effects on proliferation, migration and invasion of gastric cancer cell. Methods The expression of miRNA-101 in 28 human gastric cancer tissues, human gastric cell lines BGC-823, SGC-7901, MKN-45, AGS and human normal gastric epithelial cell line GES-1 were determined by real time polymerase chain reaction (PCR). Recombinant miRNA-101 adenovirus vector was constructed. The effects of miRNA-101 on gastric cancer proliferation was detected with cell proliferation assay. The ability of gastric cancer cell migration and invasion was assessed with Transwell assay. Gastric xenograft cancer model was established in BALB/c nude mice and the tumor size was compared. The t test was used for the statistical analysis. Results The expression of miRNA-101 in gastric cancer tissues was 0.661±0.396, which was lower than that of corresponding para carcinoma tissues (1.128±0.697), and the difference was statistically significant (t=10.091, P<0.01). The expression of miRNA-101 in normal gastric epithelial cell line GES-1 was higher than those of gastric cancer cell lines BGC-823, SGC-7901, MKN-45 and AGS. There was significant suppression role of miRNA-101 on MKN-45 cells proliferation, and which also had inhibition role on cell migration and invasion of gastric cancer cell lines BGC-823, SGC-7901, MKN-45 and AGS. At five weeks after MKN-45 gastric xenograft cancer nude mice model established, the tumor size of Ad-miRNA-101 group ((333.56±46.71) mm3) was smaller than that of Ad-enhanced green fluorescent protein (EGFP) group (806.41±51.83) mm3, and the difference was statistically significant (t=21.431, P<0.01). Conclusion In gastric tissues and cells, miRNA-101 is a tumor suppressive miRNA and its downregulated expression involved in the genesis and development of gastric cancer, which may be a new target of biological target therapy in gastric cancer. Key words: microRNAs; Stomach neoplasms; miRNA-101

MicroRNA‑34a attenuates the proliferation, invasion and metastasis of gastric cancer cells via downregulation of MET.

Proliferation, invasion and metastasis are key features of gastric cancer, contributing to high mortality rates in patients with gastric cancer worldwide. As a direct target of p53, the functions of microRNA (miR)‑34a are important, but controversial, in the progression of gastric cancer. In the present study, the clinical importance of miR‑34a in GC specimens (n=40) were investigated and were confirmed in an independent cohort from The Cancer Genome Atlas (TCGA; n=352). The prognostic value of miR‑34a was analyzed using a Kaplan‑Meier survival curve in the TCGA cohort, in combination with complete follow‑up data (n=157). The level of miR‑34a was detected in the human gastric cancer cell line and normal gastric epithelial cell line. The effect of miR‑34a on proliferation and invasion were evaluated using Cell Counting Kit 8, colony formation and cell invasion assays. The molecular basis of miR‑34a was determined by bioinformatics prediction. The correlation between miR‑34a and MET was assessed using reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The results indicated that miR‑34a was downregulated in the gastric cancer tissues, compared with the normal gastric tissues (P<0.01). miR‑34a was negatively correlated with the depth of invasion and lymph node metastasis of gastric cancer (P<0.01). In the TCGA cohort, the levels of miR‑34a were lower in T3 and T4 tumor stages, compared with the level in the T1 stage, and low levels of miR‑34a predicted significantly longer survival rates in patients with GC (P<0.05). miR‑34a also attenuated the proliferation ability, and inhibited the colony formation and cell invasion abilities of the cells (P<0.01). A negative correlation was observed between miR‑34a and MET in gastric cancer (P<0.01; r=‑0.9526), and >60% of cases exhibited consistent expression of miR‑34a and MET in gastric cancer (P<0.01). In conclusion, miR‑34a was associated with the clinicopathological features of gastric cancer and was a valuable predictor of patient prognosis. miR‑34a acted as a tumor suppressor to inhibit gastric cancer proliferation and invasion via the downregulation of MET.

Chondromodulin-1 functions as a tumor suppressor in gastric adenocarcinoma.

Chondromodulin-1 (ChM1) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. Endogenously, ChM1 is expressed in the cartilage and is an anti-angiogenic factor. ChM1 has been reported to suppress the proliferation of multiple human tumor cells in an anchorage-independent manner. However, the role of ChM1 in carcinogenesis of gastric cancer remains unknown. By quantitative RT-PCR and western blotting we examined the expression of ChM1 in gastric cancer tissue and normal gastric tissue. In vitro we investigated the functional and mechanistic roles of ChM1 in the inhibition of gastric cancer cell aggressiveness. We observed that ChM1 expression was remarkably downregulated in gastric cancer cell lines compared with the immortal normal gastric epithelial cell line GES-1. Importantly, ChM1 was frequently downregulated in gastric cancer tissue compared with normal gastric tissue. Low ChM1 mRNA expression was associated with higher clinical stages, higher lymph node metastasis, and poorer prognosis of patients. Functional assays in vitro showed that ectopic expression of ChM1 was able to inhibit gastric tumor cell proliferation by arresting the cell cycle. Overall, our findings indicate that ChM1 is a potential tumor suppressor in gastric cancer, suggesting that it may be useful as a biomarker for the treatment and prognosis of gastric cancer.

GATA-3 augmentation down-regulates Connexin43 in Helicobacter pylori associated gastric carcinogenesis

Helicobacter pylori (H. pylori) is a risk factor of gastric carcinoma, and inflammation with H.pylori infection has widely been suggested to trigger gastric carcinogenesis through “inflammation-carcinoma chain” (non-atrophic gastritis (NAG) → chronic atrophic gastritis (CAG) → intestinal metaplasia (IM) → dysplasia (DYS) and gastric carcinoma (GC)). Connexin43 (Cx43) is a major constituent of gap junction in normal gastric mucosa (NGM) and it is continuously down-regulated from normal gastric mucosa to precancerous lesions or ultimate gastric carcinoma, which shows novel target against gastric carcinoma by preventing the Cx43 decline. Our previous studies demonstrated that H. pylori infection in gastric mucosa down-regulates Cx43 expression, but its mechanism remains unknown. The transcriptional factor, GATA binding protein 3 (GATA-3) is the key to regulate adaptive immune response, which possibly relates to inflammation toward malignant transformation. Here the substantial rising of GATA-3 was screened by transcriptional factor microarray along the developmental stages of H. pylori associated gastric carcinoma. Moreover, the increased GATA-3 and inhibited Cx43 were confirmed in clinical specimens, Mongolian gerbils and normal gastric epithelial cell line GES-1 with H. pylori infection. GATA-3 silencing generated the Cx43 restoration both in intermediate differentiation gastric cancer cells BGC-803 and in H. pylori infected GES-1 cells. Dual-luciferase reporter assay further revealed the GATA-3 as one of Cx43 down-regulators by directly binding to its promoters. Together, the incremental GATA-3 is found in H. pylori associated gastric carcinogenesis, which is responsible for Cx43 inhibition as well.

421 Development and Validation of a NBI Classification System for the Prediction of Dysplasia in Barrett's Esophagus (BE): Consensus Results From an International Working Group

Background/Purpose: Visceral hypersensitivity for acid, extension and temperature stimuli in stomach and duodenum are involved in functional dyspepsia (FD). While the involvement of TRPV1 and TRPA1 have been reported in a number of animal experiments, data remains conflicting. On the other hand, ATP-gated ion channels, especially P2X3 receptor have recently been worthy of attention for its role in visceral hypersensitivity. We have reported that TRPV4 is expressed in esophageal, gastric and intestinal epithelia (J Physiol 2011, DDW2013, Am J Physiol 2013) and that ATP exocytosis is induced by TRPV4 activation in esophageal keratinocytes. We hypothesized that various physiological stimuli induced ATP release from gastric and duodenal (intestinal) epithelia through each different receptor and that excessive ATP release was involved in the pathophysiology of visceral hypersensitivity. METHODS: TRPV4 expression was examined in normal rat gastric epithelial cell line (RGE), human gastric cancer cell line (AGS) and rat intestinal cell line (IEC-6) by RT-PCR, immunostaining or Western blotting. ATP releases responding to various physiological stimuli (chemicals, low osmotic pressure, acid, extension, temperature) from each cell line were examined using luciferin-luciferase reaction. Acidic (pH 4.0), warm (40 °C) and standard (25 °C, pH7.4) solutions were used as acidic, warm stimuli or a control, respectively. Stretch stimuli was applied to cells cultured on a silicon chamber using a stretching apparatus (STREX Inc, Osaka, Japan). RESULTS: TRPV4 was expressed in RGE, IEC-6 but not in AGS. Specific TRPV4 agonist (GSK1016790A) or endogenous TRPV4 activator (5,6-EET) significantly enhanced ATP release from RGE cells but not AGS. Low osmotic, acidic, stretch or temperature stimuli significantly enhanced ATP release in RGE or IEC-6 cells. Stretchinduced ATP release from RGE cells was inhibited by pretreatment with a specific TRPV4 inhibitor, HC067047. Cell viability after each stimulus was confirmed by Trypan blue staining. Conclusion: Gastric and duodenal (intestinal) epithelial cells release ATP responding various physiological stimuli partially via TRPV4 activation . Excessive ATP release from epithelial cells might be involved in the pathophysiology of visceral hypersensitivity.

Su1826 Pseudokinase Tribbles 3 Enhances SHP2-Dependent ERK Signaling in Helicobacter pylori-Induced Gastric Carcinogenesis

Background & Aim: Helicobacter pylori (H. pylori) infection is known to induce gastric inflammation, gastric intestinal metaplasia and gastric carcinogenesis, but the details are still ambiguous. In DDW2012, we reported that H. pylori infection induced the expression of pseudokinase Tribbles 3 (Trib3) both in vitro and in vivo, and overexpression of this pesudokinase led to augmented phosphorylation of tyrosine phosphatase SHP2. In this study, we aimed to identify the downstream signaling pathway of SHP2 affected by Trib3, which contributes to gastric carcinogenesis.Methods & Results: First, we generated Trib3 overexpressing cell line Trib3GSM06 from normal murine gastric epithelial cell line GSM06. We then infected these cells with H. pylori and investigated the signaling pathway that is affected by the overexpression of Trib3. Western blot analysis of these cells showed that although neither β-catenin nor STAT3 was affected by over-expression of Trib3, the expression of phosphorylated ERK1/2 (p44/42 MAPK) was augmented up to 2.4 folds in Trib3GSM06 compared to GSM06 cells. To verify if the phosphorylation of ERK1/2 was the result of SHP2 activation, we assessed the effect of SHP2 inhibitor (II-B08) on ERK1/2 phosphorylation by western blot. As a result, the addition of SHP2 inhibitor diminished the expression of phosphorylated ERK1/2. In addition, MTS assay of Trib3GSM06 and GSM06 cells showed that over-expression of Trib3 enhanced cell proliferation, and this enhancement was suppressed by the addition of SHP2 inhibitor. Moreover, expression of c-Myc was enhanced up to 2.0 folds by overexpression of Trib3, and this enhancement was abolished by the addition of SHP2 inhibitor. Finally, we investigated the physical interaction between Trib3 and SHP2 in H. pylori-infected GSM06 cells by immunoprecipitation and western blotting, which clearly showed the binding of these two molecules. These findings suggested that over-expression of Trib3 activates ERK1/2 by directly binding to and activating SHP2. Conclusion: Our study demonstrated that H. pylori-induced Trib3 directly interacts with SHP2 and activates ERK1/2 signaling, leading to gastric carcinogenesis. This finding suggests Trib3 as a possible candidate of therapeutic target for gastric cancers.

420 Quantitative Analysis of Volumetric Laser Endomicroscopy of Histologically Correlated Images Potentially Identifies Early Neoplasia in Barrett's Esophagus

Background/Purpose: Visceral hypersensitivity for acid, extension and temperature stimuli in stomach and duodenum are involved in functional dyspepsia (FD). While the involvement of TRPV1 and TRPA1 have been reported in a number of animal experiments, data remains conflicting. On the other hand, ATP-gated ion channels, especially P2X3 receptor have recently been worthy of attention for its role in visceral hypersensitivity. We have reported that TRPV4 is expressed in esophageal, gastric and intestinal epithelia (J Physiol 2011, DDW2013, Am J Physiol 2013) and that ATP exocytosis is induced by TRPV4 activation in esophageal keratinocytes. We hypothesized that various physiological stimuli induced ATP release from gastric and duodenal (intestinal) epithelia through each different receptor and that excessive ATP release was involved in the pathophysiology of visceral hypersensitivity. METHODS: TRPV4 expression was examined in normal rat gastric epithelial cell line (RGE), human gastric cancer cell line (AGS) and rat intestinal cell line (IEC-6) by RT-PCR, immunostaining or Western blotting. ATP releases responding to various physiological stimuli (chemicals, low osmotic pressure, acid, extension, temperature) from each cell line were examined using luciferin-luciferase reaction. Acidic (pH 4.0), warm (40 °C) and standard (25 °C, pH7.4) solutions were used as acidic, warm stimuli or a control, respectively. Stretch stimuli was applied to cells cultured on a silicon chamber using a stretching apparatus (STREX Inc, Osaka, Japan). RESULTS: TRPV4 was expressed in RGE, IEC-6 but not in AGS. Specific TRPV4 agonist (GSK1016790A) or endogenous TRPV4 activator (5,6-EET) significantly enhanced ATP release from RGE cells but not AGS. Low osmotic, acidic, stretch or temperature stimuli significantly enhanced ATP release in RGE or IEC-6 cells. Stretchinduced ATP release from RGE cells was inhibited by pretreatment with a specific TRPV4 inhibitor, HC067047. Cell viability after each stimulus was confirmed by Trypan blue staining. Conclusion: Gastric and duodenal (intestinal) epithelial cells release ATP responding various physiological stimuli partially via TRPV4 activation . Excessive ATP release from epithelial cells might be involved in the pathophysiology of visceral hypersensitivity.