Gastric Adenocarcinoma Cell Line AGS Research Articles

Binding of YY1/CREB to an Enhancer Region Triggers Claudin 6 Expression in H. pylori LPS-Stimulated AGS Cells.

Aberrant expression of the tight junction protein claudin 6 (CLDN6) is a hallmark of gastric cancer progression. Its expression is regulated by the cAMP response element-binding protein (CREB). In gastric cancer induced by Helicobacter pylori (H. pylori) there is no information regarding what transcription factors induce/upregulate the expression of CLDN6. We aimed to identify whether CREB and Yin Yang1 (YY1) regulate the expression of CLDN6 and the site where they bind to the promoter sequence. Bioinformatics analysis, H. pylori lipopolysaccharide (LPS), YY1 and CREB silencing, Western blot, luciferase assays, and chromatin immunoprecipitation experiments were performed using the stomach gastric adenocarcinoma cell line AGS. A gen reporter assay suggested that the initial 2000 bp contains the regulatory sequence associated with CLDN6 transcription; the luciferase assay demonstrated three different regions with transcriptional activity, but the -901 to -1421 bp region displayed the maximal transcriptional activity in response to LPS. Fragment 1279-1421 showed CREB and, surprisingly, YY1 occupancy. Sequential Chromatin Immunoprecipitation (ChIP) experiments confirmed that YY1 and CREB interact in the 1279-1421 region. Our results suggest that CLDN6 expression is regulated by the binding of YY1 and CREB in the 901-1421 enhancer, in which a non-described interaction of YY1 with CREB was established in the 1279-1421 region.

A Survey of Naturally Occurring Molecules as New Endoplasmic Reticulum Stress Activators with Selective Anticancer Activity

The last century has witnessed the establishment of neoplastic disease as the second cause of death in the world. Nonetheless, the road toward desirable success rates of cancer treatments is still long and paved with uncertainty. This work aims to select natural products that act via endoplasmic reticulum (ER) stress, a known vulnerability of malignant cells, and display selective toxicity against cancer cell lines. Among an in-house chemical library, nontoxic molecules towards noncancer cells were assessed for toxicity towards cancer cells, namely the human gastric adenocarcinoma cell line AGS and the lung adenocarcinoma cell line A549. Active molecules towards at least one of these cell lines were studied in a battery of ensuing assays to clarify the involvement of ER stress and unfolded protein response (UPR) in the cytotoxic effect. Several natural products are selectively cytotoxic against malignant cells, and the effect often relies on ER stress induction. Berberine was the most promising molecule, being active against both cell models by disrupting Ca2+ homeostasis, inducing UPR target gene expression and ER-resident caspase-4 activation. Our results indicate that berberine and emodin are potential leads for the development of more potent ER stressors to be used as selective anticancer agents.

Isolation of Various Flavonoids with TRAIL Resistance-Overcoming Activity from Blumea lacera

Eighteen compounds, including fourteen flavonoids (1-14), one steroid (15), two fatty acids (16,17), and one nitrogen-containing compound (18), were isolated from the methanol extract of the whole Blumea lacera plant collected in Thailand. Compounds 1-11 and 15-17 exhibited tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance-overcoming activity. Among them, bonanzin (2) and cirsilineol (7) had particularly strong TRAIL resistance-overcoming activity, where the IC50 values against the human gastric adenocarcinoma cell line AGS in the presence of TRAIL (100 ng/mL) were 10.7 μM and 5.9 μM, respectively.

Effects of Fusobacterium nucleatum on migration and cytokines production of ags gastric adenocarcinoma cell line

Gastric cancer (GC) is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide. Helicobacter pylori (Hp) infection is an important risk factor for GC. However, the etiology of the tumor is multifactorial, since only 1-3% of infected patients develop cancer. Therefore, attention should be focused on the role of microbiota in gastric tumorigenesis since in some studies an alteration of the microbiota in GC has been shown. Fusobacterium nucleatum (Fn) has been found in biopsies of patients with GC. However, since its role is not clearly established, this study investigated the effects of Fn infection on the human gastric adenocarcinoma cell line AGS. Our results showed that Fn co-localized at level of the plasma membrane demonstrating the ability of Fn to adhere to AGS cells. In addition, increases in incubation times were associated with its intra-cellular localization with loss of the classic curved rod shape. Interestingly, Fn determined a greater capacity of cell migration compared to untreated AGS cells. Moreover, IL-4 expression significantly increased in Fn infected GC cells. Since cancer cell migration is an integral component of the metastatic process, additional studies are needed to better understand the mechanisms underlying the Fn/host interaction.

A new 1,2-diketone physalin isolated from Physalis minima and TRAIL-resistance overcoming activity of physalins.

A new 1,2-diketone physalin, physalin XII (1), and 13 known compounds were isolated from the methanol extract of Physalis minima whole plant collected in Thailand. Among them, five physalins (2-6) had tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistance overcoming activity, and physalin F (3) was the most active with an IC50 value of 0.39µM against human gastric adenocarcinoma cell line AGS in the presence of TRAIL (100ng/mL). An investigation of the TRAIL-resistance overcoming activity of physalins using western blot analysis showed that 3 promoted TRAIL-induced apoptosis by suppressing anti-apoptotic proteins c-FLIP and Bcl-2.

Thannilignan glucoside and 2-(β-glucopyranosyl)-3-isoxazolin-5-one derivative, two new compounds isolated from Terminalia bellirica.

Two new compounds, thannilignan 9-O-β-glucoside (1) and 2-(β-glucopyranosyl)-3-isoxazolin-5-one derivative (2), and seven known compounds were isolated from the methanol extract of Terminalia bellirica leaves, collected in Bangladesh. The structures of the compounds were elucidated using spectroscopic analysis. Among these isolated compounds, corilagin (3) was cytotoxic against human gastric adenocarcinoma cell line AGS at an IC50 of 20.8μM, and β-D-glucopyranose 1,3,6-trigallate (4) exhibited the ability to overcome tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance.

The cytotoxic activity of Sponges and Tunicates from Turkish Aegean Sea

The enormous bioactive components of different species from marine habitats make them attractive candidates for the discovery of new therapeutic active substances in several diseases such as cancer. Sea sponge and tunicate materials are the ideal sources of new chemotherapeutics for various cancers due to their rich metabolites. The fundamental purpose of the current study is to investigate the cytotoxic activity of methanolic crude extracts of sponges (Agelas oroides and Cladocora caespitosa) and tunicates (Ascidiella aspersa and Styela clava) collected from the Aegean Sea. The cytotoxic activity and the anti-cancer activity of the extracts was carried out by sodium 3´- [1- (diphenyl aminocarbonyl)-3,4-tetrazolium] -bis (4-methoxy 6-nitro) benzene sulfonic acid hydrate (XTT) on gastric adenocarcinoma cell line AGS, prostate cancer cell line PC-3, neuroblastoma cell line SH-SY5Y, and mouse fibroblast cell line L929. The extracts of sponge and tunicate were found to cause different cytotoxic effects on different cancer cells, largely in a concentration-dependent manner. It is found that the extracts exhibited high anti-cancer activities in neuroblastoma and prostate cancer cell lines at concentrations of 3 and 5 mg/ml. No significant cytotoxic activity was found in L929 cells. In addition, the microscopic examination of cells treated with the extracts shows that the extracts cause morphological changes with cellular rounding, which could be associated with apoptosis.

Cortactin Promotes Effective AGS Cell Scattering by Helicobacter pylori CagA, but Not Cellular Vacuolization and Apoptosis Induced by the Vacuolating Cytotoxin VacA.

Cortactin is an actin-binding protein and actin-nucleation promoting factor regulating cytoskeletal rearrangements in eukaryotes. Helicobacter pylori is a gastric pathogen that exploits cortactin to its own benefit. During infection of gastric epithelial cells, H. pylori hijacks multiple cellular signaling pathways, leading to the disruption of key cell functions. Two bacterial virulence factors play important roles in this scenario, the vacuolating cytotoxin VacA and the translocated effector protein CagA of the cag type IV secretion system (T4SS). Specifically, by overruling the phosphorylation status of cortactin, H. pylori alternates the activity of molecular interaction partners of this important protein, thereby manipulating the performance of cytoskeletal rearrangements, endosomal trafficking and cell movement. Based on shRNA knockdown and other studies, it was previously reported that VacA utilizes cortactin for its cellular uptake, intracellular travel and induction of apoptosis by a mitochondria-dependent mechanism, while CagA induces cell scattering, motility and elongation. To investigate the role of cortactin in these phenotypes in more detail, we produced a complete knockout mutant of cortactin in the gastric adenocarcinoma cell line AGS by CRISPR-Cas9. These cells were infected with H. pylori wild-type or various isogenic mutant strains. Unexpectedly, cortactin deficiency did not prevent the uptake and formation of VacA-dependent vacuoles, nor the induction of apoptosis by internalized VacA, while the induction of T4SS- and CagA-dependent AGS cell movement and elongation were strongly reduced. Thus, we provide evidence that cortactin is required for the function of internalized CagA, but not VacA.

M6A Methyltransferase 3 Promotes the Proliferation and Migration of Gastric Cancer Cells through the m6A Modification of YAP1.

Gastric cancer is the most common gastrointestinal tumor with an increasing incidence. Furthermore, advanced gastric cancer is more common, but the mechanism underlying the proliferation and metastasis of gastric cancer has not been thoroughly explored. N6-methyladenosine (m6A) methyltransferase 3 (METTL3) may be involved in the proliferation and metastasis of gastric cancer. Therefore, Yes-associated protein 1 (YAP1) in the Hippo pathway was selected as the target, and the relationship between METTL3 and the proliferation and metastasis of gastric cancer was proved through a series of experiments. This research showed that the expression of m6A and METTL3 was upregulated in human gastric cancer tissues and gastric cancer cell lines. After lentiviral transfection, METTL3 silencing in AGS (human gastric adenocarcinoma cell line AGS) and MKN-45 (human gastric cancer cell line MKN-45) gastric cancer cell lines directly inhibited the proliferation, aggressiveness, and migration of gastric cancer cells. Mechanically, the inhibition of the YAP1-TEAD signaling pathway by peptide 17 reduces m6A methylation and the total mRNA level of YAP1. It also eliminates the promoting effect of METTL3 on the proliferation and migration of gastric cancer cells. In turn, the overexpression of YAP1 eliminates the inhibitory effect of METTL3 silencing on the proliferation, migration, and invasion of gastric cancer cells. This article proved that m6A methyltransferase METTL3 promoted the proliferation and migration of gastric cancer cells through the m6A modification of YAP1.

Abstract 830: New bioinformatics workflow of genome-wide CRISPR-Cas9 knockout screens

Abstract Introduction: Genome-wide CRISPR-Cas9 based loss-of-function screens can be used to find essential genes for proliferation and survival of cancer cells. While recent studies have focused on establishing reference sets of essential and non-essential genes, correcting copy number effect and characterizing off-target effect, it lacks in-depth studies of the effects of gene abundance and sgRNAs that targeting multi-genomic loci. To fill this gap timely, we here present a bioinformatics workflow to reduce false positives in CRISPR-Cas9 screens. Description: Gastric adenocarcinoma cell line AGS was infected with CRISPR knockout library (TKOv3) at a multiplicity of infection of 0.3~0.4. We used the cells right after puromycin selection as the baseline sample, and the cells cultured for 14 days or 20 days as the negative selection samples. The sgRNA inserts were amplified by PCR and the corresponding libraries were sequenced on NextSeq 500 with a single-end 75 bp run, followed by analysis by MAGeCK. The read counts of sgRNAs were normalized by non-essential genes to reduce false positives. The RNA-seq data and copy number data were obtained by CCLE portal. To characterize sgRNAs targeting multiple-genomic loci, Bowtie was used to align sgRNA to the reference human genome (GRCh38) with no mismatch, and only the alignments followed by NGG PAM site were remained for downstream analysis. Summary: Integration of RNA-seq data with CRISPR negative screen results showed that the selection signal was noisy for the lowly expressed genes. The fraction of selected essential genes (overall FDR<0.05, absolute value of beta score >1) was as low as 0.11% among the genes with the bottom 10% expression level, while 27% among the genes with the top 10% expression level. After filtering out the lowly expressed genes (<0.06 RPKM), the selected essential genes had an FDR much closer to 0. Out of the 40 essential genes selected without filtering out lowly expressed genes, none of them was reported oncogenes in literature. To study the influences of multiple alignments of sgRNAs, we only considered the ones with perfect alignments (i.e., no mismatch) so that we can prevent it from being confounding with off-target effects caused by mismatch tolerance. Log fold changes in read counts were calculated for each sgRNA between a later time point (day 14 or 20) vs. baseline (day 0). The median log fold change significantly decreased as a function of the number of perfect alignments (p = 0.0001, Jonckheere trend test). This supports the hypothesis that a sgRNA aligned to several DNA targets will introduce multiple double stranded cuts, and thus will result in biased essentiality scores. Conclusions: Filtering out lowly-expressed genes prior to CRISPR screen data analysis can reduce false positives. In addition, multiple-target sgRNAs can lead to false positives but the effect needs further analysis in a case by case manner. Citation Format: Yue Zhao, Xue Wu, Yuru Wang, Kin Fai Au, Lijun Cheng, Lang Li. New bioinformatics workflow of genome-wide CRISPR-Cas9 knockout screens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 830.

Resveratrol synergizes with cisplatin in antineoplastic effects against AGS gastric cancer cells by inducing endoplasmic reticulum stress‑mediated apoptosis and G2/M phase arrest.

Gastric cancer (GC) is a common gastrointestinal malignancy, and cisplatin (DDP) is an important component of chemotherapeutic regimens for GC. However, the application of DDP is limited by its dose-dependent systemic toxicity. Resveratrol (RES) is a natural polyphenol compound that has chemopreventive and therapeutic effects against various cancers, including GC. However, whether RES can sensitize GC cells to DDP remains unknown. Following RES/DDP combination treatment, cell viability was determined by Cell Counting Kit-8 and colony-forming assays, and cell apoptosis and the cell cycle were detected by FITC-Annexin V/PI staining assay and PI staining assay, respectively, followed by flow cytometry. Moreover, western blotting was performed to evaluate the protein expression levels, and the intracellular free Ca2+ concentration was determined by a Fluo-4 AM probe after cell cotreatment with RES and DDP. The present results demonstrated that RES/DDP combination treatment significantly inhibited cell viability, promoted cell apoptosis and induced G2/M phase arrest in AGS cells. In addition, it was determined that RES combined with DDP significantly increased the levels of Bax, cleaved poly-ADP-ribose polymerase (PARP), glucose-regulated protein 78 (GRP78), PRKR-like ER kinase (PERK), p-eukaryotic translation initiation factor 2α (p-eIF2α), CCAAT/enhancer binding protein homologous protein (CHOP) and cleaved caspase-12, whereas Bcl-2 expression was downregulated following RES/DDP cotreatment. Moreover, RES/DDP cotreatment significantly upregulated phosphorylated cyclin-dependent kinase 1 (p-CDK1, Tyr15), p21Waf1/Cip1 and p27Kip1 protein levels and downregulated Cdc25C protein levels. In conclusion, RES and DDP synergistically inhibited the growth of the gastric adenocarcinoma cell line AGS by inducing endoplasmic reticulum stress-mediated apoptosis and G2/M phase arrest via activation of the PERK/eIF2α/activating transcription factor 4 (ATF4)/CHOP signaling pathway and caspase-12 and by inactivating the CDK1-cyclin B1 complex. These results indicated that RES is a promising adjuvant for DDP during GC chemotherapy.

The Helicobacter pylori Urease Virulence Factor Is Required for the Induction of Hypoxia-Induced Factor-1α in Gastric Cells

Chronic Helicobacter pylori infection increases the risk of gastric cancer and induction of hypoxia-induced factor (HIF), which is frequently associated with the development and progression of several types of cancer. We recently showed that H. pylori activation of the PI3K-AKT-mTOR pathway in gastric cells increased HIF-1α expression. Here, we identified the H. pylori virulence factor responsible for HIF-1α induction. A mutant of the H. pylori 84-183 strain was identified with reduced ability to induce HIF-1α. Coomassie blue staining of extracts from these bacteria separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed poor expression of urease subunits that correlated with reduced urease activity. This finding was confirmed in the 26695 strain, where urease mutants were unable to induce HIF-1α expression. Of note, HIF-1α induction was also observed in the presence of the urease inhibitor acetohydroxamic acid at concentrations (of 20 mM) that abrogated urease activity in bacterial culture supernatants, suggesting that enzymatic activity of the urease is not required for HIF-1α induction. Finally, the pre-incubation of the human gastric adenocarcinoma cell line AGS with blocking antibodies against Toll-like receptor-2 (TLR2), but not TLR4, prevented HIF-1α induction. In summary, these results reveal a hitherto unexpected role for the urease protein in HIF-1α induction via TLR2 activation following H. pylori infection of gastric cells.

Selective and Apoptotic Action of Ethanol Extract of Annona cherimola Seeds against Human Stomach Gastric Adenocarcinoma Cell Line AGS.

Annona cherimola is a tree belonging to the family Annonacea, whose fruit (cherimoya) is very desirable, but its seeds are considered waste. Present in these seeds are compounds that have been described as selective antiproliferative agents for cancer cells. The aim of this study was to evaluate the antiproliferative activity of ethanol macerate extract (EMCHS) obtained from A. cherimola seeds against the human stomach gastric adenocarcinoma (AGS) cell line and the normal human gastric epithelial cell line (GES-1). The EMCHS extract presented an IC50 of 80.43μg/mL in AGS cells, and a selectivity index (SI) of 3.5-fold higher than that of cisplatin. In addition, the EMCHS extract showed apoptotic activity in AGS cells since 50μg/mL. Overxpression of PUMA gene in both cells demonstrate that EMCHS activate the apoptotic route. Future studies should be carried out to elucidate anticancer activity of EMCHS in vivo. This work represents the first showing antiproliferative effects of crude extracts obtained from seeds of A. cherimola in AGS cells.

Novel RNA aptamers targeting gastrointestinal cancer biomarkers CEA, CA50 and CA72-4 with superior affinity and specificity.

Gastric cancer is the third most common cause of death from cancer in the world and it remains difficult to cure in Western countries, primarily because most patients present with advanced disease. Currently, CEA, CA50 and CA72-4 are commonly used as tumor markers for gastric cancer by immunoassays. However, the drawback and conundrum of immunoassay are the unceasing problem in standardization of quality of antibodies and time/effort for the intensive production. Therefore, there is an urgent need for the development of a standardized assay to detect gastric cancer at the early stage. Aptamers are DNA or RNA oligonucleotides with structural domain which recognize ligands such as proteins with superior affinity and specificity when compared to antibodies. In this study, SELEX (Systematic Evolution of Ligands by Exponential enrichment) technique was adopted to screen a random 30mer RNA library for aptamers targeting CEA, CA50 and CA72-4 respectively. Combined with high-throughput sequencing, we identified 6 aptamers which specifically target for these three biomarkers of gastrointestinal cancer. Intriguingly, the predicted secondary structures of RNA aptamers from each antigen showed significant structural similarity, suggesting the structural recognition between the aptamers and the antigens. Moreover, we determined the dissociation constants of all the aptamers to their corresponding antigens by fluorescence spectroscopy, which further demonstrated high affinities between the aptamers and the antigens. In addition, immunostaining of gastric adenocarcinoma cell line AGS using CEA Aptamer probe showed positive fluorescent signal which proves the potential of the aptamer as a detection tool for gastric cancer. Furthermore, substantially decreased cell viability and growth were observed when human colorectal cell line LS-174T was transfected with each individual aptamers. Taking together, these novel RNA aptamers targeting gastrointestinal cancer biomarker CEA, CA50 and CA72-4 will aid further development and standardization of clinical diagnostic method with better sensitivity and specificity, and potentially future therapeutics development of gastric cancer.

MicroRNA-25 Inhibits Cell Apoptosis of Human Gastric Adenocarcinoma Cell Line AGS via Regulating CCNE1 and MYC.

BackgroundGastric carcinoma is the second leading cause of cancer death. microRNAs play vital roles in regulating expression of related oncogenes. microRNA-25 (miR-25) has been found to be up-regulated in gastric carcinoma. However, its roles in affecting cell apoptosis of gastric carcinoma and the related mechanism remain elusive. This study aimed to uncover the influences of miR-25 on gastric carcinoma cell apoptosis and the possible functional mechanisms involved.Material/MethodsHuman gastric adenocarcinoma cell line AGS was used and transfected with lentivirus containing miR-25-specifc inhibitor sponge or expression vector to analyze the effects of miR-25.ResultsmiR-25 had higher expression in AGS than in human gastric epithelial cell line GES-1 (P<0.01). Inhibition of miR-25 by its sponge in AGS cells resulted in suppressed cell viability (P<0.01) and promoted cell apoptosis (P<0.01), while overexpression of miR-25 abrogated these effects (P<0.01 and P<0.05), indicating that miR-25 can promote cell viability and inhibit cell apoptosis in AGS cells. Expression analysis of related factors by Western blot showed that inhibiting miR-25 led to the up-regulation of F-box and WD repeat domain-containing 7 (FBXW7, P<0.01) and the down-regulation of FBXW7 substrates, cyclin E1 (CCNE1, P<0.01), and v-myc avian myelocytomatosis viral oncogene homolog (MYC, P<0.001).ConclusionsThese results indicate that miR-25 has anti-apoptosis roles in AGS cells, possibly via inhibiting FBXW7 and thus promoting oncogenes, such as CCNE1 and MYC. This study provides basic evidence for using miR-25 as a possible therapeutic target in treating gastric carcinoma.

Coiled coil rich proteins (Ccrp) influence molecular pathogenicity of Helicobacter pylori.

Pathogenicity of the human pathogen Helicobacter pylori relies on its capacity to adapt to a hostile environment and to escape the host response. Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its contribution to virulence. In this study we have explored the influence of coiled coil rich proteins (Ccrp) cytoskeletal elements on pathogenicity factors of H. pylori. Deletion of any of the ccrp resulted in a strongly decreased activity of the main pathogenicity factor urease. We further investigated their role using in vitro co-culture experiments with the human gastric adenocarcinoma cell line AGS modeling H. pylori - host cell interactions. Intriguingly, host cell showed only a weak “scattering/hummingbird” phenotype, in which host cells are transformed from a uniform polygonal shape into a severely elongated state characterized by the formation of needle-like projections, after co-incubation with any ccrp deletion mutant. Furthermore, co-incubation with the ccrp59 mutant resulted in reduced type IV secretion system associated activities, e.g. IL-8 production and CagA translocation/phosphorylation. Thus, in addition to their role in maintaining the helical cell shape of H. pylori Ccrp proteins influence many cellular processes and are thereby crucial for the virulence of this human pathogen.

Zinc ions upregulate the hormone gastrin via an E-box motif in the proximal gastrin promoter

Gastrin and its precursors act as growth factors for the normal and neoplastic gastrointestinal mucosa. As the hypoxia mimetic cobalt chloride upregulates the gastrin gene, the effect of other metal ions on gastrin promoter activity was investigated. Gastrin mRNA was measured by real-time PCR, gastrin peptides by RIA, and gastrin promoter activity by dual-luciferase reporter assay. Exposure to Zn(2)(+) ions increased gastrin mRNA concentrations in the human gastric adenocarcinoma cell line AGS in a dose-dependent manner, with a maximum stimulation of 55 ± 14-fold at 100 μM (P<0.05). Significant stimulation was also observed with Cd(2)(+) and Cu(2)(+), but not with Ca(2)(+), Mg(2)(+), Ni(2)(+), or Fe(3)(+) ions. Activation of MAPK and phosphatidylinositol 3-kinase pathways is necessary but not sufficient for gastrin induction by Zn(2)(+). Deletional mutation of the gastrin promoter identified an 11 bp DNA sequence, which contained an E-box motif, as necessary for Zn(2)(+)-dependent gastrin induction. The fact that E-box binding transcription factors play a crucial role in the epithelial-mesenchymal transition (EMT), together with our observation that Zn(2)(+) ions upregulate the gastrin gene in AGS cells by an E-box-dependent mechanism, suggests that Zn(2)(+) ions may induce an EMT, and that gastrin may be involved in the transition.

Claudin-6, 7, or 9 Overexpression in the Human Gastric Adenocarcinoma Cell Line AGS Increases Its Invasiveness, Migration, and Proliferation Rate

Altered claudin expression is related to metastatic potential, poor prognosis, or tumor recurrence. We analyzed if the overexpression of claudin-6, claudin-7, or claudin-9 in AGS cells altered cell motility, invasiveness, or proliferation rate. Claudin-7, claudin-9, and claudin-6 enhanced their invasive potential by 3.4-fold, 1.6-fold, and 2.0-fold, respectively. Claudin-6 and claudin-9 enhanced cell migration, while the proliferation rate of claudin-6-, claudin-7-, and claudin-9-transfected cells increased by 12.7%, 9.0%, and 13.3%, respectively. Claudin-7 and claudin-9 overexpression increased claudin-1 and zonula occludens-1 levels. In summary, individual increased expression of claudin-6, claudin-7, or claudin-9 is sufficient to enhance tumorigenic properties of a gastric adenocarcinoma cell line.

Green and Black Tea Inhibit Cytokine-Induced Il-8 Production and Secretion in AGS Gastric Cancer Cells via Inhibition of NF-κB Activity

Consumption of tea is associated with a reduced risk for several gastrointestinal cancers. Inflammatory processes, such as secretion of IL-8 from the gastric epithelium in response to chronic chemokine or antigen exposure, serve both as a chemoattractant for white blood cells and a prerequisite for gastric carcinogenesis. In this study, the gastric adenocarcinoma cell line AGS was used to investigate the effect of green tea extract, black tea extract, and epigallocatechin gallate (EGCG), the most abundant catechin in tea, on cytokine-induced inflammation. AGS cells were stimulated with interleukin-1β (IL-1β) to initiate inflammation, followed by exposure to either tea extracts or EGCG. We found that both green and black tea extracts at concentrations of 20 and 2 µM total catechins, respectively, significantly (p < 0.05) inhibited IL-1β-induced IL-8 production and secretion to a similar extent. Treatment of AGS cells with EGCG (8 µM) produced similar reductions in IL-1β-induced IL-8 production and secretion. Inhibition of NF-κB activity was found to be responsible, in part, for these observed effects. Our findings demonstrate that both green and black tea extracts with distinctly different catechin profiles, are capable of disrupting the molecular link between inflammation and carcinogenesis via inhibition of NF-κB activity in AGS cells.

Lysophosphatidic acid inhibits ghrelin secretion in the human gastric adenocarcinoma AGS cell line − role of mitogenic activated protein kinase signaling pathway

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor type 1a (GHS-R1a), is a 28 amino acid residue with a post-translational octanoyl modification on Ser3. Despite the biomedical interest in this hormone, the fine details of its regulation and the mechanisms controlling its secretion are largely unknown. The present study analyzes the molecular steps involved in the full lysophosphatidic acid (LPA) receptor-mediated activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and its consequent role as an inhibitor of ghrelin secretion in the gastric adenocarcinoma cell line AGS. ERK1/2 phosphorylation mediated by LPA proceeds via activation of the type 2 LPA receptor, activation of the nonreceptor tyrosine kinase c-Src, and subsequent transactivation of the epidermal growth factor receptor. Furthermore, LPA-induced ERK activation was found to be independent of matrix metalloproteinases; thus, c-Src acted as the scaffold-transactivating epidermal growth factor receptor. Finally, a correlation was observed between the mitogenic effects of LPA and ghrelin secretion in the human gastric adenocarcinoma cell line AGS. These data suggest a possible physiological role of LPA in ghrelin secretion. The relationship found between LPA and ghrelin secretion might explain the low circulating levels of ghrelin observed in obese patients, as a bona fide reflex of the energetic stores.