Human Gastric Mucosal Cell Line Research Articles

Exploring the Mechanism of KLF15 on the Biological Activity and Autophagy of Gastric Cancer Cells based on PI3K/Akt/Mtor Signaling Pathway.

To explore the mechanism of KLF15 on the biological activity and autophagy of gastric cancer cells based on the PI3K/Akt/mTOR signaling pathway. The gastric cancer AGS cells were divided into the Con group, pcDNANC group, pcDNA-KLF15 group, LY294002 group and IGF-1 group. RT-PCR was used to detect the expression of KLF15 in human gastric mucosal cells and gastric cancer cells; MTT method to detect cell proliferation; Transwell method to detect cell invasion; flow cytometry to detect cell apoptosis; Western blotting to detect PI3K, Akt, mTOR in cells, LC3, Beclin1, p62 protein expression.P<0.05 was used to indicate statistical significance. Compared with the human gastric mucosal cell line GES-1 cells, the expression of KLF15 in human gastric cancer cell lines MKN-28, MFC, SCG-7901 and AGS cells was significantly decreased, And the expression of KLF15 in AGS cells, was the lowest (P=0.006). Compared with the Con group, The expression of KLF15 in the cells of the PCDNA-KLF15 group was significantly increased (P=0.018); There was no significant difference in the expression of KLF15 between the Con group and the PCDNA-NC group (P=0.225). Compared with the Con group, the proliferation and invasion abilities of the cells in the pcDNA-KLF15 group were significantly reduced, And the apoptosis ability was significantly increased (P=0.019). The ratio of LC3II/LC31 and the expression of Beclin1 Protein in the control group were significantly higher than those in the Con group (P=0.017). Overexpression of KLF15 can inhibit the proliferation and invasion of Gastric cancer cells and promote cell apoptosis and autophagy, and its mechanism may be related to the regulation of the PI3K/Akt/mTOR signaling pathway.

Synthesis of Conformationally Liberated Yohimbine Analogues and Evaluation of Cytotoxic Activity.

A modular synthetic approach to strategically unique structural analogues of the alkaloid yohimbine is reported. The overall synthetic strategy couples the transition-metal-catalyzed decarboxylative allylation of 2,2-diphenylglycinate imino esters with a scandium triflate-mediated highly endo-selective intramolecular Diels–Alder (IMDA) cycloaddition to generate a small collection of de-rigidified yohimbine analogues lacking the ethylene linkage between the indole and decahydroisoquinoline units. One compound generated in this study contains an unprecedented pentacyclic urea core and appears to demonstrate increased cytotoxicity against the gastric cancer cell line SGC-7901 in comparison to a pancreatic cancer cell line (PATU-8988) and a normal human gastric mucosal cell line (GES-1).

Synthesis and anticancer evaluation of novel 1H-benzo[d]imidazole derivatives of dehydroabietic acid as PI3Kα inhibitors.

Phosphatidylinositol 3-kinase (PI3K) is one of the most attractive therapeutic targets for cancer treatment. In this study, a series of new 2-arylthio- and 2-arylamino-1H-benzo[d]imidazole derivatives of dehydroabietic acid were designed, synthesized and characterized by 1H NMR, 13C NMR, IR and MS spectra analyses. In the in vitro anticancer assay, some title compounds showed significant inhibitory activities against four cancer cell lines (HCT-116, MCF-7, HeLa and HepG2). Among them, compound 9g exhibited the most potent activity with IC50 values of 0.18±0.03, 0.43±0.05, 0.71±0.08 and 0.63±0.09μM against four cancer cell lines, and considerably lower cytotoxicity to human gastric mucosal cell line Ges-1 (IC50: 21.95±0.73μM). Besides, compound 9g displayed a certain selective activity to PI3Kα (IC50=0.012±0.002μM) over PI3Kβ, γ and δ, and meanwhile, it can remarkably decrease the expression level of p-Akt (Ser473). In addition, compound 9g could increase intracellular reactive oxygen species level, decrease mitochondrial membrane potential, upregulate Bax and cleaved caspase-3/9 levels, downregulate Bcl-2 level and thus induce the apoptosis of HCT-116 cells in a dose-dependent manner. The results suggested that compound 9g could be considered as a promising PI3Kα inhibitor.

Eukaryotic translation initiation factor 3B downregulation inhibits cell proliferation and promotes cell apoptosis through negatively regulating tumor necrosis factor receptor superfamily member 21 in gastric cancer

Background: This study aimed to detect eukaryotic translation initiation factor 3B (EIF3B) expression in gastric cancer (GC) cell lines, and further explore the effect of EIF3B downregulation on GC cell proliferation and apoptosis. Methods: EIF3B mRNA expression and protein expression in human GC cell lines (NCI-N87, AGS, HGC-27, BGC-823 and MGC80-3) and human gastric mucosal epithelial cell line (GES-1) were detected. Control siRNA (Si-NC group) and EIF3B siRNA (Si-EIF3B group) were transfected into NCI-N87 cells. Rescue experiment was performed by transfection of EIF3B siRNA (Si-EIF3B group) and EIF3B siRNA plus tumor necrosis factor receptor superfamily member 21 (TNFRSF21) siRNA (Si-EIF3B & Si-TNFRSF21 group) into NCI-N87 cells. Besides, cell proliferation, apoptosis, TNFRSF21 expression and TRAF1 expression were assessed. Results: EIF3B mRNA expression and protein expression were elevated in NCI-N87, AGS, HGC-27 and BGC-823 cell lines compared to GES-1 cell line. In NCI-N87 cells, proliferation was reduced in Si-EIF3B group compared to Si-NC group. For cell apoptosis, its rate and apoptotic marker C-Caspase 3 expression were increased but anti-apoptosis marker Bcl-2 expression was reduced in Si-EIF3B group compared to Si-NC group. Moreover, mRNA expression and protein expression of TNFRSF21 were increased in Si-EIF3B group compared to Si-NC group, while mRNA expression and protein expression of TRAF1 were reduced in Si-EIF3B group compared to Si-NC group. In rescue experiment, cell proliferation was increased but apoptosis was decreased in Si-EIF3B & Si-TNFRSF21 group compared to Si-EIF3B group. Conclusions: EIF3B is overexpressed in GC cell lines, and its downregulation inhibits cell proliferation while promotes apoptosis through negatively regulating TNFRSF21 in GC.

Synthesis and antiproliferative activity of novel 4-azasteroidal-17-hydrazone derivatives

A new series of 4-azasteroidal-17-hydrazone derivatives have been synthesized from androstenedione. Their structures were characterized by analysis and spectroscopic data. The antiproliferative activity of synthesized compounds against three cancer cells (human lung adenocarcinoma, human oesophageal cervical cancer, human gastric adenocarcinoma) and a normal cell line (human gastric mucosal) was investigated. The studies show that the compound bearing a naphthyl group displayed the same antiproliferative activity in vitro against tested cells as cis-platin did (a positive control). Most of the compounds show very weak toxicity towards normal human gastric mucosal cell line.

Up-Regulation of MiR-1915 Inhibits Proliferation, Invasion, and Migration of Helicobacter pylori-Infected Gastric Cancer Cells via Targeting RAGE.

PurposeHelicobacter pylori (HP)-infected gastric cancer (GC) is known to be a fatal malignant tumor, but the molecular mechanisms underlying its proliferation, invasion, and migration remain far from being completely understood. Our aim in this study was to explore miR-1915 expression and its molecular mechanisms in regulating proliferation, invasion, and migration of HP-infected GC cells.Materials and MethodsQuantitative real-time PCR and western blot analysis were performed to determine miR-1915 and receptor for advanced glycation end product (RAGE) expression in HP-infected GC tissues and gastritis tissues, as well as human gastric mucosal cell line GES-1 and human GC cell lines SGC-7901 and MKN45. CCK8 assay and transwell assay were performed to detect the proliferation, invasion, and migration capabilities. MiR-1915 mimics and miR-1915 inhibitor were transfected into GC cells to determine the target relationship between miR-1915 and RAGE.ResultsMiR-1915 was under-expressed, while RAGE was over-expressed in HP-infected GC tissues and GC cells. Over-expressed miR-1915 could attenuate cellular proliferation, invasion, and migration capacities. RAGE was confirmed to be the target gene of miR-1915 by bioinformatics analysis and luciferase reporter assay. Moreover, HP-infected GC cellular proliferation, invasion, and migration were inhibited after treatment with pcDNA-RAGE.ConclusionMiR-1915 exerted tumor-suppressive effects on cellular proliferation, invasion, and migration of HP-infected GC cells via targeting RAGE, which provided an innovative target candidate for treatment of HP-infected GC.

MicroRNA-190 regulates FOXP2 genes in human gastric cancer

ObjectiveTo investigate how microRNA-190 (miR-190) regulates FOXP2 genes in gastric cancer (GC) cell line SGC7901.MethodsWe identified that miR-190 could target FOXP2 genes by using dual luciferase enzyme assay. Precursor fragment transfection of miR-190 was performed with GC cell line SGC7901 and human gastric mucosal cell line GES-1. miR-190 expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and FOXP2 protein expression was measured by Western blotting.ResultsFOXP2-3′-untranslated region (UTR) in miR-190 transfection group was significantly decreased as compared with other groups. There were no significant differences in fluorescence signals of FOXP2mut-3′-UTR in each group. Therefore, it was assumed that miR-190 can target FOXP2 genes. Through RT-PCR verification, it was observed that the expression level of miR-190 was significantly higher in GC cell line SGC7901 than in human gastric mucosa cell line GES-1 after transfection with miR-190 mimics. The expression level of miR-190 was significantly higher in GES-1 cells than in SGC7901 cells after transfection with miR-190 inhibitors. Western blotting results showed the expression level of FOXP2 was significantly lower in GC cell line SGC7901 than in GES-1 cells. Compared with blank, mimics control, and inhibitors control groups, the miR-190 mimics group showed significantly enhanced proliferation, migration, and invasion abilities, while miR-190 inhibitors group showed decreased abilities toward proliferation, migration, and invasion (P<0.05). The transcription level of miR-190 and the expression level of FOXP2 in tumor tissues and adjacent normal tissues in GC patients were verified to be consistent with those of cell line experiments.ConclusionUpregulation of miR-190 can lead to downregulation of FOXP2 protein expression. miR-190 may serve as a potential target for GC diagnosis.

Role of heat shock proteins in oxygen radical–induced gastric apoptosis

BackgroundThe generation of reactive oxygen species (ROS) and their resultant oxidative damage is a common pathway for gastric mucosal injury. Developing strategies to protect the gastric epithelium against oxygen free radical damage is of profound pathophysiological interest. We have previously shown caspase-mediated apoptosis as a major cause of ROS-induced cell death in gastric mucosa. Because heat shock proteins (Hsps) confer protection against many cytotoxic agents, this study was undertaken to determine whether modulation of Hsps was protective against oxidative damage. Materials and methodsAGS cells (human gastric mucosal cell line) received either no pretreatment, heat shock pretreatment (1 h at 42 ± 1°C), or pretreatment with an Hsp modulating drug (geldanamycin or quercetin). Cells were then exposed to hydrogen peroxide (H2O2), a representative ROS (1 mM, a physiologically relevant concentration), for 24 h. Caspase-3 activation and Poly ADP Ribose Polymerase (PARP) inactivation, as well as DNA-histone complex formation were used as measures of apoptosis. Inducible Hsps (Hsp70 and Hsp90) were detected using Western blot analysis. ResultsResults showed heat shock pretreatment induced increased expression of Hsp70 without change in Hsp90. In response to H2O2 exposure alone, there was significant increase in DNA-histone complex formation as well as caspase-3 activation and PARP cleavage in gastric epithelium. Heat shock pretreatment resulted in statistically significant prevention in these measures of apoptosis. Geldanamycin increased Hsp70, but elicited cleavage of Hsp90 and subsequently resulted in an increase in H2O2-induced apoptosis. Quercetin decreased Hsp70 and resulted again in increased H2O2-induced apoptosis. ConclusionsThese findings indicate that heat shock pretreatment protects gastric mucosal cells against H2O2-induced apoptosis and that Hsp70 and Hsp90 may play key roles in this process. These results further suggest that perturbations in Hsp metabolism may induce mucosal injury in response to oxygen free radicals.

Anti- Helicobacter pylori activity of crude acetonic extracts of Streptomyces sp. H7372

IntroductionHelicobacter pylori is a gram negative bacterium and a gastric pathogen that infects approximately 50% of the world's populations. Prolonged H. pylori infection causes chronic inflammation and significantly increases the risk of developing gastric cancer, making it a serious global health issue. Recent studies have showed that the success of current treatment, the standard triple therapy, using a wide range of antibiotics (amoxicillin, metronidazole, tetracycline etc.), has declined to unacceptable rates of 80% or less due to antibiotic resistance. New perspectives at current anti- Helicobacter pylori therapy and strategies as well as novel drug alternatives are essentially needed to eradicate H. pylori infection. ObjectiveWe reported a new strain Streptomyces sp. H7372, a gram-positive aerobic bacterium isolated from mangrove soil in Sabah, Malaysia that has shown anti-Helicobacter pylori activities. MethodsStreptomyces sp. H7372 was extracted once with acetone and dried at 40°C. The dried extract was dissolved in distilled water and was examined in vitro. Broth microdilution method was used to determine minimum inhibitory concentration of Streptomyces sp. H7372 against H. pylori ATCC 700824 strain. For disc assay, both ATCC and some clinical strains of H. pylori were grown on TSA blood agar with varying extract concentrations to study zone of inhibition. ResultsThe acetonic extract was found to be fairly inhibitory to Helicobacter pylori ATCC 700824 strain (MIC: 0.0625 ug/ul) as compared to Amoxicillin (MIC: 1×10−5 ug/ul). Disc assay showed concentration dependent inhibitory effect. ConclusionThe present study shows that the acetone extracts from Streptomyces sp. H7372 displayed a strong potential alternative against H. pylori. In the future, extract will be successively fractioned into different chemical portions and will be examined in vitro. Anti-inflammation and anti-adhesion assays will be performed in human gastric mucosal cell line infected with H. pylori to understand the mechanisms of anti-H.pylori activity of H7372.

Identification of microRNAs associated with lymphangiogenesis in human gastric cancer

Lymphatic metastasis is a primary cause of gastric cancer-related death, yet factors governing tumor cell lymphatic metastasis have not been fully elucidated. MicroRNAs (miRNAs) are a recently discovered class of regulatory, non-coding RNAs, some of which are involved in gastric cancer progression. However, little is known about which miRNA contributes to the lymphatic metastasis in human gastric cancer. This prompted us to find the significant miRNAs associated with lymphangiogenesis in human gastric cancer. We screened vascular endothelial growth factor C (VEGF-C) expression in several gastric cancer cell lines as well as in the immortalized human gastric mucosal cell line GES-1, by real-time reverse transcriptase PCR (qRT-PCR). The gastric cancer cell lines MKN-45 and SGC-7901, which have commonly been cultured with human lymphatic endothelial cells (HLECs) in vitro, promoted tube formation of HLECs following transformation with a VEGF-C expression vector. Using microarrays, we identified a panel of differentially expressed miRNAs in HLECs that had been co-cultured with VEGF-C-transformed gastric cancer cells compared with non-transformed gastric cancer cells. A subset of miRNAs was further validated using qRT-PCR. We found altered expression of miRNAs in HLECs co-cultured with lymphangiogenesis-inducing VEGF-C-transformed gastric cancer cells, with 47 up-regulated and 42 down-regulated miRNAs. These findings were confirmed by qRT-PCR of selected miRNAs. Furthermore, several miRNAs were differentially expressed in patients with positive lymphatic metastasis of the primary gastric tumor. Up-regulated miRNAs included miR-648, miR-5002-3p, miR-4754, miR-4760-5p, miR-4491, miR-4252, miR-5007-3p, and miR-647; and down-regulated miRNAs included miR-3178, miR-593-5p, miR-4485, miR-135a-3p, miR-17, miR-1469, and miR-124-5p. Several lymphangiogenesis-related miRNAs are significantly altered during lymphatic metastasis of gastric cancer.

In vitro anti-Helicobacter pylori activity of a flavonoid rich extract of Glycyrrhiza glabra and its probable mechanisms of action

Ethnopharmacological relevanceGlycyrrhiza glabra Linn. is regarded useful for peptic ulcer in traditional systems of medicine in India and Helicobacter pylori has been considered as one of the causative factors for peptic ulcer. Aim of the present study is to evaluate the anti-Helicobacter pylori action of GutGard®, a flavonoid rich extract of Glycyrrhiza glabra and further to elucidate the possible mechanisms of its anti-Helicobacter pylori action. Materials and methodsAgar dilution and microbroth dilution methods were used to determine the minimum inhibitory concentration of GutGard® against Helicobacter pylori. Protein synthesis, DNA gyrase, dihydrofolate reductase assays and anti-adhesion assay in human gastric mucosal cell line were performed to understand the mechanisms of anti-Helicobacter pylori activity of GutGard®. ResultsGutGard® exhibited anti-Helicobacter pylori activity in both agar dilution and microbroth dilution methods. Glabridin, the major flavonoid present in GutGard® exhibited superior activity against Helicobacter pylori while glycyrrhizin did not show activity even at 250μg/ml concentration. In protein synthesis assay, GutGard® showed a significant time dependent inhibition as witnessed by the reduction in 35S methionine incorporation into Helicobacter pylori ATCC 700392 strain. Additionally, GutGard® showed a potent inhibitory effect on DNA gyrase and dihydrofolate reductase with IC50 value of 4.40μg/ml and 3.33μg/ml respectively. However, the extract did not show significant inhibition on the adhesion of Helicobacter pylori to human gastric mucosal cell line at the tested concentrations. ConclusionThe present study shows that, GutGard® acts against Helicobacter pylori possibly by inhibiting protein synthesis, DNA gyrase and dihydrofolate reductase.

Gastric mucosal cell model for estimating relative gastrointestinal toxicity of non-steroidal anti-inflammatory drugs

The study objective was to characterize the AGS human gastric mucosal cell line as a model for estimating gastrointestinal toxicity of COX-inhibiting compounds. Rofecoxib, celecoxib, nimesulide, ibuprofen, indomethacin, aspirin, salicylic acid, naproxen and acetaminophen were tested for inhibition of COX-2-mediated prostaglandin E 2 synthesis in A549 and AGS cells. The IC 50 ratio AGS/A549 was calculated as an estimate of the therapeutic index (TI) for gastrointestinal toxicity. Calculated IC 50 values of non-steroidal anti-inflammatory drugs (NSAIDs) in A549 cells were in excellent agreement with published values ( r=0.996; P<0.005). Calcium ionophore induction of arachidonic acid release in AGS cells provided TI similar to those using platelets and A549 cells ( r=0.918; P<0.01). The AGS/A549 model exhibited lower TI than the platelet/A549 model. Spearman ranking correlated clinical NSAID gastropathy with lower AGS TI values. The AGS cell line has excellent potential to serve as a model for assessing the gastrointestinal effects of COX-inhibiting compounds.

Role of calcium homeostasis in gastric mucosal injury and protection

Using a human gastric mucosal cell line, known as AGS cells, we determined the role that perturbations in intracellular Ca 2+ concentration [Ca 2+] i might play in cellular injury induced by various damaging agents. For deoxycholate (CD) and ethanol (EtOH) induced damage, a concentration related increase in [Ca 2+] i was noted that preceded and closely paralleled the magnitude of injury. Thus, the higher the concentration of DC or EtOH, the more profound were the changes in [Ca 2+] i and the resultant degree of cellular injury. Pretreatment with a low concentration of DC (50 μM; called a mild irritant) that was not damaging by itself attenuated injury induced by a damaging concentration (i.e. 250 μM) of DC, and appeared to elicit this protective action through mechanisms that resisted intracellular Ca 2+ accumulation. Additional studies indicated that the mechanism of aspirin damage may be similar and that other protective agents such as prostaglandins and growth factors appear to mediate their protective properties through prevention of intracellular Ca 2+ alterations. We propose that agents that prevent mucosal injury mediate this activity through a cellular response (involving active Ca 2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca 2+ accumulation.