Revealing physical properties of gastric adenocarcinoma cells with two distinct morphologies linking to preferential cellular migration and proliferation.

Studies have found that miR-665 acted as a tumor suppressor or an oncogene in different malignancies. miR-665 expression was elevated in gastric adenocarcinoma tissues; however, its role and mechanism in this disease are not fully clarified. The expression of miR-665 and its target gene was detected in human gastric adenocarcinoma tissues and cells. Moreover, we analyzed the effects of miR-665 on the proliferation, migration, and epithelial-mesenchymal transition (EMT) of gastric adenocarcinoma cells as well as tumor growth in vivo. The mechanisms of miR-665 in gastric adenocarcinoma were investigated by using molecular biology techniques. We found miR-665 was upregulated and suppressor of cytokine signaling 3 (SOCS3) was downregulated in gastric adenocarcinoma tissues and cells. Elevated miR-665 was positively correlated with tumor size, lymph node metastasis, invasion depth, TNM stage, and poor differentiation in gastric adenocarcinoma patients. Overexpression of miR-665 promoted, whereas knockdown of miR-665 suppressed the proliferation, migration, and EMT of gastric adenocarcinoma cells. Furthermore, we demonstrated that miR-665 functioned through targeting SOCS3, followed by activation of the FAK/Src signaling pathway in gastric adenocarcinoma cells. miR-665 antagomir inhibited tumor growth as well as the activation of the FAK/Src pathway but increased SOCS3 expression in nude mice. In addition, miR-665 expression was negatively regulated by long noncoding RNA maternally expressed gene 3 (MEG3). In conclusion, miR-665 acted as an oncogene in gastric adenocarcinoma by inhibiting SOCS3 followed by activation of the FAK/Src pathway and it was negatively modulated by MEG3. miR-665 may be a promising therapeutic target for the treatment of gastric adenocarcinoma.

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulcer disease and distal gastric adenocarcinoma, a process for which adherence of H. pylori to gastric epithelial cells is critical. Decay-accelerating factor (DAF), a protein that protects epithelial cells from complement-mediated lysis, also functions as a receptor for several microbial pathogens. In this study, we investigated whether H. pylori utilizes DAF as a receptor and the role of DAF within H. pylori-infected gastric mucosa. In vitro studies showed that H. pylori adhered avidly to Chinese hamster ovary cells expressing human DAF but not to vector controls. In H. pylori, disruption of the virulence factors vacA, cagA, and cagE did not alter adherence, but deletion of DAF complement control protein (CCP) domains 1-4 or the heavily O-glycosylated serine-threonine-rich COOH-terminal domain reduced binding. In cultured gastric epithelial cells, H. pylori induced transcriptional up-regulation of DAF, and genetic deficiency of DAF attenuated the development of inflammation among H. pylori-infected mice. These results indicate that DAF may regulate H. pylori-epithelial cell interactions relevant to pathogenesis.

Resveratrol-induced inactivation of human gastric adenocarcinoma cells through a protein kinase C-mediated mechanism

FOXD2 regulations IQGAP3 mediated Ca2+ signaling pathway to facilitate gastric adenocarcinoma cell promotion

Bacterial Pathogen Helicobacter pylori: A Bad AKTor Inhibits p53 Protein Activity [corrected

Gastric cancer is the fifth most common malignancy worldwide. Serjania marginata Casar. (SM) displays anti-inflammatory properties and has been used to treat gastrointestinal disorders. In the current study, we examined whether the hydroethanolic extract of SM leaves exerted cytotoxic, mutagenic, and protective effects in non-tumor gastric epithelium cells (MNP01) and gastric adenocarcinoma cells (ACP02) in vitro and analyzed whether its action was selective. Initially, cell viability (MTT assay), cell cycle kinetics (flow cytometry), and cell proliferation (total protein content) were analyzed. In addition, genomic instability (cytokinesis-block micronucleus cytome assay), anti/pro-oxidant status (CM-H2 DCFDA probe), and transcriptional expression (RT-qPCR) of genes related to cell cycle, cell death, and antioxidant defense were also evaluated. The SM extract was cytotoxic toward MNP01 and ACP02 cells at concentrations greater than 300 and 100 μg·ml-1 , respectively, and decreased protein content only toward ACP02 cells at 200 μg ml-1 . In ACP02 cells, the SM extract at 100 μg·ml-1 associated with doxorubicin (DXR; 0.2μg ml-1 ) clearly promoted cell cycle arrest at the G2/M phase. The extract alone was not mutagenic to either cell type and reversed DXR-induced DNA damage and H2 O2 -induced oxidative stress in MNP01 cells. The gene expression experiments showed that SM hydroethanolic extract exerts an antioxidant response via NFE2L2 activation in non-tumor gastric cells, and cell cycle arrest (G2/M) in ACP02 gastric cancer cells via the TP53 pathway. The selective action of SM indicates that it is a promising therapeutic agent to treat gastric diseases and merits further studies.

The phthalate plasticizer, di(2-ethyl-hexyl) phthalate (DEHP), and its derived metabolites are common anthropogenic environmental toxins, which are known to act as endocrine disruptors. Numerous studies have associated DEHP with disruption of sex hormones, abnormal development of reproductive organs, allergies, and inflammation. Its role in promoting inflammation has been reported by both human epidemiological and animal studies. In stomach tissue, chronic inflammation is known to accompany mucosal damage, and pave the way to gastritis, stomach ulcers, and ultimately gastric cancer. Eastern Asian populations possess the highest gastric cancer incidences in the world. Coincidentally, East Asia is one of the world's major sites for plastics manufacture and export. Thus, possible correlations between DEHP, a common plasticizer, and gastric cancer are of great interest. Our study revealed several critical findings. First, even at very low dosage, mimicking the residual plasticizer exposure, detrimental effects of DEHP on gastric cells can be detected. Second, gastric cells treated with DEHP increased cyclooxygenase-2 (COX-2) in a time-dependent manner. Third, promoter deletion studies revealed a critical role of nuclear factor-kappa B (NF-κB) for COX-2 gene responses. Finally, our results indicated that a low concentration of DEHP is able to trigger COX-2 activation via the extracellular signal-regulated kinase (ERK1/2) and NF-κB signaling pathway. Taken together, we demonstrate that very low doses of DEHP enhance the expression of the prototypical inflammatory gene, COX-2, in gastric cancer cells via ERK1/2 and NF-κB activation. This study provides important insights into the inflammatory process and damages associated with phthalate plasticizers exposure.

Tolectin is a non-steroid anti-inflammatory drug (NSAID) which has a clinically analgesic and antipyretic activity. Although the mechanism of its action is not known, tolectin inhibits prostaglandin synthetase in vitro and lowers the plasma level of prostaglandin E in humans. Recently, the anticancer effects of tolectin has been reported in in vitro studies. The aim of this study was to investigate the in vitro tolectin effects on antitumor TRP53 gene expression level in gastric adenocarcinoma cells. The gastric adenocarcinoma (AGS) cells were divided to control group and groups treated with 0.0012, 0.0023, 0.0049, 0.0097, 0.0194, 0.0389 mmol/mL. MTT assay method was used to determine the cell viability. Relative TRP53 gene expression level was evaluated by quantitative Real Time PCR. Data were analyzed using ANOVA and student’s t-test. Cell viability significantly decreased in all groups of tolectin treated AGS cells compared to control group; however, expression level of the TRP53 gene did not significantly changed in cells treated with effective concentration of tolectin (0.0131 mmol/mL) compared with control group. Our findings indicated that tolectin has a significant cytotoxic effects on gastric adenocarcinoma cells, however, its effective cytotoxic concentration has not significant impact on antitumor TRP53 gene expression level.

The microRNA-200 (miR-200) family has been reported to induce epithelial differentiation and suppress epithelial-mesenchymal transition (EMT) by inhibiting translation of zinc finger E-box-binding homeobox (ZEB) 1 and 2 mRNAs in several types of cancers. This study aimed to clarify the role of miR-200b in regulating EMT and promoting cellular proliferation, invasion, and migration in gastric cancer. The relationships among the expression levels of miR-200b, ZEB1 and ZEB2, and E-cadherin mRNAs were analyzed by quantitative real-time reverse transcription-polymerase chain reaction in frozen tissue samples from 40 gastric cancer patients who underwent gastrectomy from 2008 to 2010. The effects of miR-200b on EMT in gastric cancer cells in vitro were also analyzed. Diffuse histologic type, depth of tumor, tumor size, lymph node metastasis, and lymphatic invasion were significantly higher in the low-miR-200b expression group compared with the high expression group. There was a strong correlation between the levels of miR-200b, and ZEB2 and E-cadherin mRNAs in gastric cancer patients. Upregulation of miR-200b in gastric cancer cells changed the cell morphology from fibroblast- to epithelial-like, associated with localization of E-cadherin to the plasma membrane. ZEB2 mRNA levels fell, while E-cadherin expression levels increased in gastric cells overexpressing miR-200b, associated with significantly reduced cellular proliferation, and inhibition of cellular migration and invasion. miR-200b regulates ZEB2 expression and thus controls metastasis in gastric cancer.

It has been proposed that the expression of Fas ligand (Fas L) in tumors may play an important role in immune escape. This study was undertaken to test a 'counterattack' theory as a mechanism of immune escape in gastric carcinoma. Expression of Fas and Fas L was examined in the human gastric cancer cell lines using reverse transcription-polymerase chain reaction. Cytotoxicity was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Apoptosis of target Jurkat cells was examined after coculture with the effector gastric cancer cells in vitro. Immunohistochemical staining was performed for the detection of Fas and FasL in tumor-infiltrating lymphocytes (TIL) and gastric cancer cells in vivo. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method in vitro and in vivo. Fas and FasL mRNA were found to be differentially expressed in gastric cancer cell lines. The coculture experiment showed that apoptosis of Jurkat was induced by a FasL-overexpressing effector gastric cell SNU-484. In a Fas-expressing gastric cell SNU-638, Fas expression was upregulated by the treatment of gamma-interferon in a time- and concentration-dependent manner. SNU-638 treated with gamma-interferon was more sensitive to anti-Fas antibody-mediated cytotoxicity than was the control cell line, suggesting an increase of functional Fas in gastric cancer cells. The expression of FasL in gastric cancer cells and of Fas in apoptotic TIL was also detected in vivo. The data indicate that the FasL expression of gastric cancer cells supports a 'counterattack theory' in gastric cancer cells and that the upregulation of Fas by IFN-gamma in SNU-638 may accelerate the apoptosis pathway through the Fas and FasL interaction between gastric cancer cells and immune cells. This result is supported by the expression of FasL in gastric cancer cells and apoptotic TIL in vivo. It is implicated that the different biological behaviors of gastric cancer cells could be at least in part explained by Fas and FasL interaction with immune cells.

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been reported in some cancer cells, including AGS human gastric adenocarcinoma cells. Reducing this resistance might shed light on the treatment of human gastric adenocarcinoma. In this study, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors can restore TRAIL responsiveness in gastric adenocarcinoma cells. The effect of two GSK-3 inhibitors, SB-415286, and LiCl, on apoptosis signaling of TRAIL in human gastric adenocarcinoma cell lines and primary gastric epithelial cells was analyzed. Both inhibitors can sensitize gastric adenocarcinoma cells, but not primary gastric epithelial cells, to TRAIL-induced apoptosis by increasing caspase-8 activity and its downstream signal transmission. Adding p53 siRNA can downregulate GSK-3 inhibitor-related sensitization to TRAIL-induced apoptosis and caspase-3 activity. GSK-3 inhibitors strongly activate the phosphorylation of JNK. Inhibition of JNK leads to earlier and more intense apoptosis, showing that the activation of JNK may provide anti-apoptotic equilibrium of pro-apoptotic cells. Our observations indicate that GSK-3 inhibitors can sentize AGS gastric adenocarcinoma cells to TRAIL-induced apoptosis. Therefore, in certain types of gastric adenocarcinoma, GSK-3 inhibitor might enhance the antitumor activity of TRAIL and mightbe a promising candidate for the treatment of certain types of gastric adenocarcinoma.

Background: The Wnt signaling pathway is required for proliferation of intestinal stem cells and activating mutations in the Wnt pathway can promote colorectal cancer. The requirement for Wnt to support gastric stem cell function is much less clear, despite recent reports showing that activating Wnt pathway mutations occur in human gastric cancer. Here we tested the role of Wnt to support stem cell proliferation in the mouse stomach and in human stem cells cultured from control or gastric cancer tissues. Methods: Wnt signaling in the mouse stomach was detected using TCF/Lef:H2B-GFP reporter mice, and Wnt+ gastric cells were identified via GFP immunostaining. Wnt function in SOX2+ gastric stem cells was tested with Sox2-CreER; Apcfl/+ mice, 1-month post-Apc deletion. Wnt regulation of human gastric stem cells was tested in organoid cultures derived from paired control (non-cancer) and gastric cancer tissues. Established organoids were treated with the Wnt inhibitor IWR-1-endo and growth was assessed by quantification of organoid size. Results: GFP+ cells were observed in the gastric epithelium of TCF/Lef:H2B-GFP mice, located throughout corpus glands and at the gland base in antral glands. Interestingly, GFP+ epithelial cells were largely post-mitotic although in the antrum, a subset of GFP+ cells at the gland base co-localized with the antral stem cell marker LGR5. In both corpus and antrum, a subset of GFP+ epithelial cells co-stained with the pan-endocrine marker Chromogranin A and in the antrum, a subset also co-stained with the deep mucous cell marker GSII, suggesting Wnt may regulate gastric cell fate. Analysis of Sox2-CreER; Apcfl/+ mice revealed that Wnt pathway activation in SOX2+ gastric stem cells led to increased epithelial cell proliferation in both corpus and antrum. In the antrum, this incease in proliferation was also accompanied by increased expression of the cancer stem cell marker CD44 at the gland base. Wnt activation also perturbed epithelial cell differentiation in the gastric antrum, with expansion of GSII+ deep mucous cells. Wnt inhibition reduced growth of human gastric organoids derived from non-cancer and gastric cancer tissues. Interestingly, organoids derived from gastric cancer tissue were significantly larger than control organoids and required a 5-fold increase in IWR-1-endo to achieve significant growth inhibition, implying that gastric cancer organoids are somewhat resistant to Wnt inhibition to slow growth. Conclusion: Wnt signaling is a critical pathway for regulation of mouse and human gastric stem cell homeostasis, promoting cellular proliferation. Thus, Wnt may represent an effective therapeutic target for gastric cancers with Wnt-activating mutations. Citation Format: Elise Susan Demitrack, Linda C. Samuelson. Wnt signaling promotes mouse and human gastric stem cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 913. doi:10.1158/1538-7445.AM2017-913

Introduction: Apoptosis has provided solutions for effective anticancer treatments, and today one of the most interesting strategies is drug interventions that can mediate the death of cancer cells through the induction of apoptosis. Many plant compounds are known to have anti-tumor activity in this way. Compound Honey Syrup is used in Iranian traditional medicine and its cytotoxicity has been determined on melanoma cells. The purpose of this research was to investigate the anticancer effect of Compound Honey Syrup herbal product on gastric adenocarcinoma cells and the mechanism of this effect. Method: The lethal effect of Compound Honey Syrup on gastric adenocarcinoma (AGS) and fibroblastic (HgF) cells after 24 hours of incubation was investigated by MTT calorimetric method. Annexin FITC-V and PI staining method determined apoptotic cell death. The change in the activity of caspases 8 and 9 was also evaluated by enzymatic method. The base membrane of the model was used to evaluate the invasion ability of Compound Honey Syrup cancer cells. Results: Compound Honey Syrup had a strong and concentration-dependent killing effect on AGS cells and caused cell death mainly by inducing early apoptosis. The increased activity of caspases 8 and 9 was involved in this process. Compound Honey Syrup also reduced the ability of cancer cells to invade. Conclusion: By activating caspases 8 and 9, COMPOUND HONEY SYRUP led to the apoptotic death of human gastric cancer cells. In addition, COMPOUND HONEY SYRUP can be an effective factor for investigating the prevention or treatment of stomach cancer in humans by reducing the strength and invasion of cancer cells.

Protein kinase C (PKC) family members are multifunctional kinases that have been implicated in many cell biological and physiological tasks including acid, pepsinogen, and mucous production. Through the use of small-molecule PKC modulators, PKC has been found to be involved in gene expression, the control of cytoskeleton, membrane and secretagogue-dependent signal transduction for secretion of acid. Gastric carcinoma and adenocarcinoma cells often show dysregulated PKC-dependent cell signal transduction compared to normal gastric cells. Moreover, PKC was the first known target of tumor promoting phorbol esters. These findings support PKC as a potential chemotherapy target in gastric cancer. Various approaches have been launched in directly targeting PKC for chemotherapy of gastric cancer. The macrocyclic lactone bryostatin-1 is a promising agent that acts as a modulator of PKC activity, and enhances the effect of chemotherapeutic agents such as paclitaxel. This article provides an overview of the findings to date regarding the physiological role of PKC in the gastric cell system by various pharmacological approaches and examines PKC as a target in gastric (adeno-)carcinoma chemotherapy.

At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.