Gastric Epithelial Cell Line Research Articles

α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the gastric mucosa and triggers various stomach diseases. H. pylori induces reactive oxygen species (ROS) production and DNA damage. The heterodimeric Ku70/Ku80 protein plays an essential role in the repair of DNA double-strand breaks (DSB). Oxidative stress stimulate apoptosis and DNA damage that can be repaired by Ku70/80. However, excessive reactive oxygen species (ROS) can cause Ku protein degradation, resulting in DNA fragmentation and apoptosis. α-lipoic acid (α-LA), which is found in organ meats such as liver and heart, spinach, broccoli, and potatoes, quenches free radicals, chelates metal ions, and reduces intracellular DNA damage induced by oxidative stress. Here, we investigated whether H. pylori decreases Ku70/80 and induces apoptosis, and whether α-LA inhibits changes induced by H. pylori. We analyzed ROS, DNA damage markers (γ-H2AX, DNA fragmentation), levels of Ku70/80, Ku–DNA binding activity, Ku80 ubiquitination, apoptosis indices (Bcl-2, Bax, apoptosis-inducing factor (AIF), and caspase-3), and viability in a human gastric epithelial adenocarcinoma cell line (AGS). H. pylori increased ROS, DNA damage markers, Ku80 ubiquitination, and consequently induced apoptosis. It also decreased nuclear Ku70/80 levels and Ku–DNA-binding activity; increased Bax expression, caspase-3 cleavage, and truncated AIF; but decreased Bcl-2 expression. These H. pylori-induced alterations were inhibited by α-LA. The antioxidant N-acetylcysteine and proteasome inhibitor MG-132 suppressed H. pylori-induced cell death and decreased nuclear Ku70/80 levels. The results show that oxidative stress induced Ku70/80 degradation via the ubiquitin–proteasome system, leading to its nuclear loss and apoptosis in H. pylori-infected cells. In conclusion, α-LA inhibited apoptosis induced by H. pylori by reducing ROS levels and suppressing the loss of Ku70/80 proteins in AGS cells.

Helicobacter pylori Thioredoxin1 May Play a Highly Pathogenic Role via the IL6/STAT3 Pathway.

Background Recent studies have shown that CagA is considered highly pathogenic to helicobacter pylori (HP) in Western populations. However, in East Asia, CagA positive HP can be up to 90%, but not all patients will lead to gastric cancer. Our research group has found that HP thioredoxin1 (Trx1) may be a marker of high pathogenicity. Here, we investigate whether HP Trx1 exerts high pathogenicity and its internal molecular mechanism. Materials and Methods We constructed the coculture system of high-Trx1 HP and low-Trx1 HP strains with gastric epithelial cell lines separately and detected the influence of HP strains. The cells were stained by AM/PI, and the cell's mortality was assessed by fluorescence microscope. The cell's supernatants or precipitates were collected to detect the expression of IL6. In addition, the cell's precipitates were collected, and the expression of p-STAT3 was detected by western blot. Furthermore, the cell's supernatants were collected for detecting the expression of 8-OHDG to investigate the extent of DNA damage. Results The high-Trx1 HP can cause higher mortality of GES-1 cells compared with the low-Trx1 HP group (high-Trx1 HP (4.53 ± 0.56) %, low-Trx1 HP (0.39 ± 0.10) %, P < 0.001). The mRNA and protein level of IL-6 in AGS and GES-1 cells were increased during HP infection, and the expression of IL-6 in the High-Trx1 HP group was much higher than the low-Trx1 HP group. Besides, the expression of p-STAT3 was higher in the HP-positive gastric mucosa. And the expression of p-STAT3 in the high-Trx1 HP group was significantly upregulated compared with the low-Trx1 HP group. Furthermore, the expression of 8-OHDG in the high-Trx1 group was much higher than the low-Trx1 group (high-Trx1 HP (5.47 ± 1.73) ng/ml, low-Trx1 HP (2.89 ± 1.72) ng/ml, P < 0.05). Conclusion HP Trx1 may play as a marker of high pathogenicity, and the high-Trx1 HP could mediate the pathogenic process of HP infection via the IL6/STAT3 pathway.

Vacuolating Cytotoxin A Triggers Mitophagy in Helicobacter pylori-Infected Human Gastric Epithelium Cells.

Helicobacter pylori (H. pylori)-derived vacuolating cytotoxin A (VacA) causes damage to various organelles, including mitochondria, and induces autophagy and cell death. However, it is unknown whether VacA-induced mitochondrial damage can develop into mitophagy. In this study, we found that H. pylori, H. pylori culture filtrate (HPCF), and VacA could activate autophagy in a gastric epithelial cell line (GES-1). VacA-caused mitochondrial depolarization retards the import of PINK1 into the damaged mitochondria and evokes mitophagy. And, among mass spectrometry (LC-MS/MS) identified 25 mitochondrial proteins bound with VacA, Tom20, Tom40, and Tom70, TOM complexes responsible for PINK1 import, were further identified as having the ability to bind VacA in vitro using pull-down assay, co-immunoprecipitation, and protein–protein docking. Additionally, we found that the cell membrane protein STOM and the mitochondrial inner membrane protein PGAM5 also interacted with VacA. These findings suggest that VacA captured by STOM forms endosomes to enter cells and target mitochondria. Then, VacA is transported into the mitochondrial membrane space through the TOM complexes, and PGAM5 aids in inserting VacA into the inner mitochondrial membrane to destroy the membrane potential, which promotes PINK1 accumulation and Parkin recruitment to induce mitophagy. This study helps us understand VacA entering mitochondria to induce the mitophagy process.

High Expression of UPK3A Promotes the Progression of Gastric Cancer Cells by Inactivating p53 Pathway.

Background Gastric cancer is a common gastrointestinal tract cancer and is a considerable health burden worldwide. TCGA analysis found Uroplakin 3A (UPK3A) was upregulated in gastric cancer tissues. Our study was designed to investigate the underlying mechanism of Uroplakin 3A (UPK3A) in gastric cancer. Methods Data from TCGA database were used to assess the expression, and Kaplan-Meier plotter analysis was used to assess the prognosis value of UPK3A. Furthermore, there are effects of UPK3A silencing on the activity, proliferation, migration, and invasion of human gastric cancer cells (SNU-216 and HGC-27) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, wound healing, and Transwell assays. In addition, the expression of UPK3A, p53, KLF4, ZMAT3, MDM2, and SP1 was detected by qRT-PCR and Western blot assay. Results UPK3A was markedly upregulated in gastric cancer tissues compared to that in normal tissues, and patients with high UPK3A level showed poor prognosis. UPK3A was highly expressed in human gastric cancer cell lines compared to that in a normal human gastric epithelial cell line. Silencing of UPK3A inhibited the proliferation, migration, and invasion of gastric cancer cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that UPK3A was involved in the p53 signaling pathway. UPK3A suppressed the activation of p53 signaling pathway, and treatment with Pifithrin-α (an inhibitor of the p53 signaling pathway) or silencing of p53 significantly reversed the effect of UPK3A silencing on the expression of p53, KLF4, ZMAT3, MDM2, and SP1. Conclusion Our findings showed that UPK3A promotes the progression of gastric cancer by regulating the p53 signaling pathway and could be a potential therapeutic target for gastric cancer.

Astaxanthin Inhibits Helicobacter pylori-Induced MMPs Expression and Cell Invasion by Suppressing PI3K-AKT-mTOR Pathways in AGS Cells

ObjectivesMatrix metalloproteinases (MMPs) are members of the metzincin group of proteases which can degrade various components of extracellular matrix components (ECM). ECM degradation by MMPs not only enhances tumor invasion but also affects tumor cell behavior and leads to cancer progression. Resent reports demonstrated that PI3K-AKT-mTOR signaling pathways are involved in expression of MMPs. Especially when it comes to gastric cancer, H. pylori infection promotes the invasion and metastasis of tumor cells by reactive oxygen species (ROS). Astaxanthin, a xanthophyll carotenoid, has been well known for its strong antioxidant and anticancer properties. Thus, the present study was aimed to investigate the inhibitory effects and mechanisms of astaxanthin on H. pylori-induced MMPs expression and cell invasion in human gastric epithelial cells.MethodsHuman gastric epithelial cell lines, AGS were used. AGS cells were pre-treated with astaxanthin for 3 hours prior to H. pylori (cag A positive NCTC 11,637 strains) infection at bacterium/cell ratio of 50:1 and cultured for 24 hours in the presence of H. pylori. mRNA expression and protein levels of MMP-7 and 10 was measured by real time PCR and Western blot analysis, respectively. Intracellular ROS levels were determined using DCF-DA. Invasive cell phenotype was measured using invasion assay.ResultsAstaxanthin inhibited expression of MMP-7&10. Intracellular ROS production and cell invasion induced by H. pylori was inhibited by astaxanthin. In addition, H. pylori-induced activation of PI3K-AKT-mTOR was inhibited by astaxanthin.ConclusionsAstaxanthin exerts an inhibitory effect on H. pylori-induced MMPs expression and cell invasion by suppression of ROS production and PI3K-AKT-mTOR signaling pathway in gastric epithelial cells. Thus astaxanthin may hold promise for clinical cancer therapy.Funding SourcesNo funding sources.

Interaction mechanism between ZnO nanoparticles‐whey protein and its effect on toxicity in GES‐1 cells

The interaction between zinc oxide nanoparticles (ZnO NPs) and whey protein (WP) was studied. The gastric epithelial cell line (GES-1) was used to evaluate the toxicity intensity of ZnO NPs. The interaction mechanism of ZnO NPs and WP was studied by spectroscopic techniques. The results showed that the inhibitory effect of ZnO NPs on cells activity could be reduced when added to ZnO NPs at a concentration of 50µg/ml. The fluorescence quenching mechanism of ZnO NPs on WP is a combination of dynamic and static quenching. The interaction force between ZnO NPs and WP can be considered as H-bond and VdW force, and they have two binding sites. The interaction between WP and ZnO NPs leads to the loosening of the structural skeleton of WP and the extension of peptide chain, which exposes the tyrosine (Tyr) and tryptophan (Trp) hydrophobic groups in the hydrophobic region of protein molecules and reduces the hydrophobicity of the microenvironment. The ZnO NPs might form a complex with WP through H-bond, hydrophobic interactions, and so on, leading to peptide chain rearrangement, and finally causing changes in the secondary structure of α-helix. Practical Application This study provides a theoretical basis for future research on the interaction between food ingredients and nanomaterials, the evaluation of toxicity of nanomaterials and the application scope of nanomaterials in food field.

MiR-1273h-5p suppresses CXCL12 expression and inhibits gastric cancer cell invasion and metastasis.

The aim of this study was to verify the biological function of miR-1273h-5p in gastric cancer (GC) and its underlying mechanisms. The differential expression of microRNAs between GC and tumor-adjacent normal tissues was detected using microarrays, miR-1273h-5p, and chemokine (C-X-C motif) ligand 12 (CXCL12) mRNA, and protein levels were evaluated using polymerase chain reaction and Western blotting methods, cell proliferation, apoptosis, migration, and invasion were determined by CCK-8, flow cytometry, and transwell assay. Compared to tumor-adjacent normal tissue and gastric epithelial mucosa cell line cells, miR-1273h-5p was significantly downregulated in tissues and cells of GC. The overexpression of miR-1273h-5p could inhibit cell proliferation, migration, invasion, and promote cell apoptosis; in contrast, inhibition of miR-1273h-5p expression could reverse this process. Moreover, a significant upregulation of CXCL12 was observed when the miR-1273h-5p was downregulated in GC cells. Additionally, miR-1273h-5p significantly reduces tumor volume and weight. Thus, this study suggests that miR-1273h-5p regulates cell proliferation, migration, invasion, and apoptosis during GC progression by directly binding to CXCL12 mRNA 3′-untranslational regions, which may be a novel diagnostic and therapeutic target in GC.

Biodegradation of Fumonisins by the Consecutive Action of a Fusion Enzyme.

Fumonisins (FBs) are toxic mycotoxins that commonly exist in food and feed. FBs can induce many aspects of toxicity, leading to adverse effects on human and animal health; therefore, investigating methods to reduce fumonisin contamination is necessary. In our study, we generated a recombinant fusion enzyme called FUMDI by linking the carboxylesterase gene (fumD) and the aminotransferase gene (fumI) by overlapping polymerase chain reaction (PCR). The fusion enzyme FUMDI was successfully, secretively expressed in the host Pichia pastoris (P. pastoris) GS115, and its expression was optimized. Our results demonstrated that the fusion enzyme FUMDI had high biodegradation activity of fumonisin B1 (FB1) and other common FBs, such as fumonisin B2 (FB2) and fumonisin B3 (FB3), and almost completely degraded 5 μg/mL of each toxin within 24 h. We also found that FUMDI enzyme and its reaction products had no negative effect on cell viability and did not induce cell apoptosis, oxidative stress, or endoplasmic reticulum (ER) stress in a human gastric epithelial cell line (GES-1). The results indicated that these FBs degradation products cannot have adverse effects in a cell model. In conclusion, a safe and efficient fumonisin-degrading enzyme was discovered, which could be a new a technical method for hazard control of FBs in the future.

Bmi-1 directly upregulates glucose transporter 1 in human gastric adenocarcinoma.

This study aimed to investigate whether and how Moloney murine leukemia virus integration site 1 (Bmi-1) plays a role in the regulation of glucose transporter 1 (GLUT1) in gastric adenocarcinoma (GAC). GAC and matched noncancerous tissues were obtained from GAC patients who underwent surgical treatment at the China-Japan Union Hospital, Jilin University (Changchun, Jilin, China). The human GAC cell line AGS and the gastric epithelial cell line GES-1 were used for in vitro studies. BALB/c nude mice were used for in vivo studies. The Bmi-1 and GLUT1 protein levels were significantly greater in human tissues from GAC patients and AGS cells in comparison with controls. Silencing of Bmi-1 resulted in significant decrease in glucose uptake, lactate levels, and GLUT1 expression. In vivo 18F-deoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) imaging studies indicated that the nude mice bearing xenografts of AGS cells treated with Bmi-1-specific small interfering RNA (siRNA) had a significantly lower maximum standardized uptake value (SUVmax) in comparison with the control mice. Thus, Bmi-1 directly upregulates GLUT1 gene expression, through which it is involved in enhancing glucose uptake in GAC. The results also provide scientific evidence for 18F-FDG PET/CT imaging to evaluate Bmi-1 and glucose uptake in GAC.

Quercetin exerts anti-inflammatory effects via inhibiting tumor necrosis factor-α-induced matrix metalloproteinase-9 expression in normal human gastric epithelial cells.

BACKGROUNDGastric injury is the most common digestive system disease worldwide and involves inflammation, which can lead to gastric ulcer or gastric cancer (GC). Matrix metallopeptidase-9 [MMP-9 (gelatinase-B)] plays an important role in inflammation and GC progression. Quercetin and quercetin-rich diets represent potential food supplements and a source of medications for treating gastric injury given their anti-inflammatory activities. However, the effects and mechanisms of action of quercetin on human chronic gastritis and whether quercetin can relieve symptoms remain unclear.AIMTo assess whether tumor necrosis factor-α (TNF-α)-induced MMP-9 expression mediates the anti-inflammatory effects of quercetin in normal human gastric mucosal epithelial cells.METHODSThe normal human gastric mucosa epithelial cell line GES-1 was used to establish a normal human gastric epithelial cell model of TNF-α-induced MMP-9 protein overexpression to evaluate the anti-inflammatory effects of quercetin. The cell counting Kit-8 assay was used to evaluate the effects of varying quercetin doses on cell viability in the normal GES-1 cell line. Cell migration was measured using Transwell assay. The expression of proto-oncogene tyrosine-protein kinase Src (c-Src), phospho (p)-c-Src, extracellular-signal-regulated kinase 2 (ERK2), p-ERK1/2, c-Fos, p-c-Fos, nuclear factor kappa B (NF-κB/p65), and p-p65 and the effects of their inhibitors were examined using Western blot analysis and measurement of luciferase activity. p65 expression was detected by immunofluorescence. MMP-9 mRNA and protein levels were measured by quantitative reverse transcription polymerase chain reaction (qRT–PCR) and gelatin zymography, respectively.RESULTSqRT-PCR and gelatin zymography showed that TNF-α induced MMP-9 mRNA and protein expression in a dose- and time-dependent manner. These effects were reduced by the pretreatment of GES-1 cells with quercetin or a TNF-α antagonist (TNFR inhibitor) in a dose- and time-dependent manner. Quercetin and TNF-α antagonists decreased the TNF-α-induced phosphorylation of c-Src, ERK1/2, c-Fos, and p65 in a dose- and time-dependent manner. Quercetin, TNF-α antagonist, PP1, U0126, and tanshinone IIA (TSIIA) reduced TNF-α-induced c-Fos phosphorylation and AP-1–Luciferase (Luc) activity in a dose- and time-dependent manner. Pretreatment with quercetin, TNF-α antagonist, PP1, U0126, or Bay 11-7082 reduced TNF-α-induced p65 phosphorylation and translocation and p65–Luc activity in a dose- and time-dependent manner. TNF-α significantly increased GES-1 cell migration, and these results were reduced by pretreatment with quercetin or a TNF-α antagonist.CONCLUSIONQuercetin significantly downregulates TNF-α-induced MMP-9 expression in GES-1 cells via the TNFR-c-Src–ERK1/2 and c-Fos or NF-κB pathways.

Differential H. pylori-Induced MAPK Responses Regulate Lewis Antigen Expression and Colonization Density on Gastric Epithelial Cells Between Children and Adults.

Helicobacter pylori causes gastrointestinal diseases, the manifestations of diseases are more serious in adults than in children. Lewis antigen expressions on the gastric epithelium serves as receptors targeted by H. pylori. Moreover, the MAPK signaling pathway involves glycoprotein synthesis of Lewis antigens. We aimed to investigate whether differences in H. pylori-induced MAPK responses mediate gastric Lewis antigens expression and colonization density differently in children and adults. We used human stomach fetal epithelium (HSFE) and SV40-immortalized human normal gastric epithelial (GES-1) cell lines to mimic primary gastric epithelium of children and adults, respectively. H. pylori colonization intensity and Lewis antigens were significantly higher in GES-1 than in HSFE cells, whereas IL-8 and IL-6 levels were significantly higher in HSFE than in GES-1 cells after infection. c-Jun N-terminal kinase (JNK) siRNA and inhibitor (SP600125) experiments showed that Lewis antigen expression and H. pylori colonization were reduced in GES-1 cells but increased in HSFE cells. Furthermore, p-p38 intensity was significantly higher in the superficial epithelium of the children than in the adults with/without H. pylori infection. The overexpression of p38 in GES-1 cells downregulated H. pylori-induced JNK activity mimicking H. pylori infection in children. In conclusion, a higher p38 expression in gastric epithelium counteracting JNK activity in children may contribute to lower Lewis antigen expression and colonization density than in adults after H. pylori infection.

Long non-coding RNA TTTY15 silencing inhibits gastric cancer progression by sponging microRNA-98-5p to down-regulate cyclin D2 expression

ABSTRACT Gastric cancer is the most common malignant tumor in the digestive system. However, the detection rate of early gastric cancer is low, resulting in delayed prognosis and poor outcomes. The identification of effective therapeutic targets for gastric cancer is, therefore, of profound significance. Recently, various lncRNAs have been shown to be biomarkers for different cancers. This study investigated the role of long non-coding RNA (lncRNA) TTTY15 in gastric cancer. The expression level of TTTY15, miR-98-5p, and cyclin D2 (CCND2) were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot assay using tumor and non-tumor tissues collected from 30 patients with gastric cancer, gastric cancer cell lines (AGS, SNU-5, and NCI-N87), and the normal gastric epithelial cell line GES-1. The interaction between TTTY15 and miR-98-5p and between miR-98-5p and CCND2 were predicted by bioinformatics and then further verified by dual-luciferase and RNA pull-down analyses. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) assay, and apoptosis was measured using flow cytometry and caspase-3 assay. The results indicate that TTTY15 and CCND2 expression increased and miR-98-5p expression decreased in gastric cancer tumor tissues and cell lines. TTTY15 knockdown inhibited gastric cancer cell proliferation but promoted apoptosis by sponging miR-98-5p, which acted as a tumor suppressor gene by reducing the expression of its target gene CCND2 in gastric cancer. In conclusion, lncRNA TTTY15 is a potential oncogene involved in gastric cancer and may be a novel therapeutic target for gastric cancer treatment.

MiR-153-3p Attenuates the Development of Gastric Cancer by Suppressing SphK2.

Gastric cancer (GC) is the second leading cause of cancer-related mortality worldwide. MicroRNAs (miRNAs) have been extensively reported to play a role in GC development; however, it remains unknown whether miR-153-3p participates in the nosogenesis of GC. GC tissues along with the adjacent nontumor tissues were obtained from 50 patients with GC. Moreover, we incubated human GC cell lines (SGC7901, AGS, MGC803, and BGC823) and a gastric epithelial cell line (GES-1) and then transfected BGC823 cells with miR-153-3p and DNA/SphK2 vector to determine the action of miR-153-3p and SphK2 on GC. RT-qPCR was performed to determine the levels of miR-153-3p and sphingosine kinase 2 (SphK2). The viability of BGC823 cells was measured by the CCK-8 assay, while wound healing assays and transwell assays were used to measure the migration and invasion ability of BGC823 cells. Western blotting analysis and immunohistochemistry (IHC) were conducted to evaluate the level of SphK2. The binding ability of miR-153-3p and SphK2 was determined by dual-luciferase reporter assays. The expression level of miR-153-3p was reduced in GC tissues and cells, while the SphK2 was enhanced. An increase in miR-153-3p level led to a decline in the growth and metastasis of GC cells and increased their apoptosis. Moreover, a decrease in miR-153-3p level elevated GC cells growth and metastasis, and attenuated their apoptosis. SphK2 was also corroborated as a downstream gene of miR-153-3p. Here, SphK2 expression was elevated in GC tissues and cells, indicating SphK2 might be involved in the development of GC. Rescue assays showed that miR-153-3p could reverse the effect of SphK2 on the cell growth, metastasis, and the apoptosis of GC cells. In conclusion, this study showed that miR-153-3p suppressed the growth and metastasis in GC cells by regulating SphK2, which might facilitate the search for novel biomarkers to treat GC.

Zinc finger protein 852 is essential for the proliferation, drug sensitivity, and self-renewal of gastric cancer cells.

Exploring cellular and molecular mechanisms responsible for gastric cancer growth, survival, self-renewal, and metastasis helps develop efficacious therapeutic strategies. In this study, the expression and function of zinc finger protein 852 (ZNF852) in human gastric cancer cell lines were characterized. ZNF852 was upregulated in gastric cancer cell lines relative to normal gastric epithelial cell line GES-1. When the ZNF852 gene was ablated in gastric cancer cell line MGC-803 using the CRISPR/Cas9-encoding lentivirus, the proliferation of MGC-803 was suppressed. ZNF852 deficiency also resulted in the inhibition of MGC-803 sphere formation, along with decreases in SRY-box 2 (SOX2), octamer-binding transcription factor 4 (OCT4), and Nanog homeobox (NANOG), suggesting that ZNF852 sustains self-renewal of MGC-803 cells. Furthermore, ZNF852 deficiency increased oxaliplatin-induced MGC-803 cell death, implying the role of ZNF852 in drug sensitivity. Subcutaneous infusion of MGC-803 cells into nude mice illustrated the same effects of ZNF852 on the proliferation and self-renewal of gastric cancer cells. Similar effects of ANF852 were also seen in gastric cancer cell line SNU-1. Interestingly, ZNF852 deficiency caused downregulation of epidermal growth factor receptor (EGFR) on gastric cancer cells. In summary, this study uncovers the positive regulatory role of ZNF852 in gastric cancer growth and maintenance. ZNF852 could be a potential therapeutic target for inhibiting gastric cancer initiation or progression.

Berbamine Suppresses the Growth of Gastric Cancer Cells by Inactivating the BRD4/c-MYC Signaling Pathway.

PurposeBerbamine (Ber), a bioactive constituent extracted from a traditional Chinese medicinal herb, has been shown to exhibit broad inhibitory activity on a panel of cancer cell types. However, its effects and the underlying molecular mechanisms on gastric cancer (GC) remain poorly understood.MethodsThe anti-growth activity of Ber on two GC cell lines and normal gastric epithelial cell line were evaluated using MTS and clone formation assay. Flow cytometry analysis was employed to evaluate the cell cycle distribution and apoptosis of GC cells. Western blot and quantitative PCR (qPCR) analysis were employed to investigate the anti-GC mechanism of Ber. The inhibitory activity and binding affinity of Ber against BRD4 were evaluated by homogeneous time-resolved fluorescence (HTRF) and surface plasmon resonance (SPR) assay, respectively. Molecular docking and molecular simulations were conducted to predict the interaction mode between BRD4 and Ber.ResultsThe results demonstrated that Ber reduced the proliferation of GC cell lines SGC-7901 and BGC-823 and induced cell cycle arrest and apoptosis. Mechanistically, Ber was identified as a novel natural-derived BRD4 inhibitor through multiple experimental assay, and its anti-GC activity was probably mediated by BRD4 inhibition. Molecular modeling studies suggested that Ber might bind to BRD4 primarily through hydrophobic interactions.ConclusionOur study uncovered the underlying anti-GC activity of Ber in vitro and suggested that Ber holds promise as a potential lead compound in the discovery of novel BRD4 inhibitors.

LncRNA FENDRR Inhibits Gastric Cancer Cell Proliferation and Invasion via the miR-421/SIRT3/Notch-1 Axis.

ObjectiveThis study aimed to investigate the regulatory effect of lncRNA fetal-lethal non-coding developmental regulatory RNA (FENDRR) on gastric cancer (GC) progression.MethodsThe expression levels of FENDRR in GC tissues and paracancerous tissues, as well as in gastric normal epithelial cell line and GC cell lines were detected. The Ad-FENDRR or si-FENDRR was transfected into AGS and SGC-7901 cells, and cell proliferation, invasion and apoptosis were determined. Online bioinformatics database predicted and screened miR-421 as a potential target of FENDRR, and SIRT3 was predicted as a target gene of miR-421. The pcDNA-SIRT3 or si-SIRT3 was transfected into AGS cells, and cell proliferation, invasion, apoptosis and Notch-1 protein expression were determined. Ad-FENDRR was transfected into AGS and SGC-7901 cells alone or together with miR-421 mimic to explore the effect of miR-421 on cells. The AGS cells transfected with Ad-FENDRR were injected into the armpits of nude mice to establish subcutaneous xenograft tumor model, and tumor growth was observed.ResultsFENDRR expression was downregulated in GC tissues and cell lines. Overexpression of FENDRR or SIRT3 inhibited tumor proliferation and invasion, and promoted apoptosis. The overexpression of Notch-1 reversed the inhibitory effect of SIRT3 on AGS cell. MiR-421 mimic reversed the inhibitory effect of FENDRR on the growth of AGS and SGC-7901 cells. Nude mice injected with FENDRR overexpressing AGS cells had smaller tumor volume and weight and weaker tumor cell proliferation ability.ConclusionFENDRR inhibits Notch-1 pathway to inhibit GC cell proliferation and invasion by upregulating SIRT3 expression via targeting miR-421.

Abstract LBA050: Bioenergetic evaluation of Mito-compound Mito-Met as potent cytotoxic agents in gastric cancer

Abstract Gastric cancer (GC) kills around 800,000 people each year around the world, placing it among the deadliest types of cancer. The evolution of intestinal metaplasia is unknown, but there are different factors that are involved in its development, the diet rich in salt, the Epstein-Barr virus, Helicobacter pylori and a wide variety of genes that affect cell activity. Currently, surgical resection with chemotherapy is the main treatment with a 5-year survival of 30% that varies according to the metastatic extension. The mitochondria is now one of the therapeutic targets in the development of new compounds with anticancer capacity and it has been observed that metformin behaves as an insulin sensitizer at high concentrations, inhibiting the mitochondrial complex I. For this reason, Mito-metformin (Mito-Met) was designed, this is a mitochondrial analogue of metformin that is linked to the lipophilic alkyl triphenylphosphonium cation (TPP) with a mitochondrial effect 1000 times greater at low concentrations. In this study, the effects of Mito-Met were evaluated in two GC cell lines (AGS and KATO III) and in the healthy gastric epithelium cell line (HPSEC). Cell viability, cytotoxicity, colony formation and cell migration assays were performed. Also, it was evaluated whether Mito-Met had the potential to affect mitochondrial bioenergetics by evaluating the OCR oxygen consumption rate, ECAR extracellular acidity rate, glycolytic capacity and mitochondrial fuel oxidation in live cells in real time using the analyzer XFe24 extracellular flux from Seahorse Biosciences. A dose-time-dependent decrease in cell viability was observed with IC50 values of 2.9 µM, 19,3 µM and 22,1 µM in AGS, KATO and HPSEC cells, respectively. The results in clonogenic assay showed that Mito-Met strongly affected AGS with an IC50 of 11.7 uM and a greater impact was observed in HPSEC than in KATO III, with IC50 values of 31.8 uM and 3.6 x 103 uM, respectively. The results of the wound healing assay showed that Mito-Met has a significant time-dependent dose effect on metastasis. Mito-Met affects the mitocondrial bioenergetics, our results showed affectation of basal respiration, maximum respiration, ATP production and reserve capacity in both cell lines. Mito-Met decreased baseline OCR and stressed OCR in a dose-dependent manner in both GC cell lines. Mito-Met increased the metabolic potential in a dose-dependent manner of OCR in AGS, on the contrary, a dose-dependent decrease of ECAR was observed in KATO III. There was no change in HPSEC. These results show that Mito-Met is promising compound for treatment of GC. Citation Format: Giovanni A Lineros Franco, Yenny Bueno, Micael Hardy, Olivier Ouari, Julio C. Montoya-Villegas, Marcos López-Casilla, Sandra M. Sanabria-Barrera. Bioenergetic evaluation of Mito-compound Mito-Met as potent cytotoxic agents in gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA050.

Quercetin Improves Mitochondrial Function and Inflammation in H2O2-Induced Oxidative Stress Damage in the Gastric Mucosal Epithelial Cell by Regulating the PI3K/AKT Signaling Pathway.

Functional dyspepsia (FD) is one of the most common functional gastrointestinal disorders, the therapeutic strategy of which it is limited due to its complex pathogenesis. Oxidative stress-induced damage in gastric mucosal epithelial cells is related to the pathogenesis and development of FD. Quercetin (Que) is one of the active ingredients of Zhishi that showed antioxidant, antiapoptotic, and anti-inflammatory effects. The aim of this study is to investigate the effect of Que on oxidative stress-induced gastric mucosal epithelial cells damage and its underlying molecular mechanism. The gastric mucosal epithelial cell line GES-1 was treated with 200 μM of H2O2 to construct an oxidative stress-induced damage model. The H2O2 cells were then administrated with different concentrations of Que. The results indicated that high concentration of Que (100 μM) showed cytotoxicity in H2O2-induced GES-1 cells. However, appropriate concentration of Que (25 and 50 μM) alleviated the oxidative stress damage induced by H2O2, as demonstrated by the increase of proliferation, decrease of ROS generation, apoptosis, inflammation, and alleviation of mitochondrial function and cell barrier. In addition, Que increased the activation of phosphorylation of PI3K and AKT decreased by H2O2. To investigate whether Que alleviated the oxidative stress damage in GES-1 cells by the PI3K/AKT signaling pathway, the GES-1 cells were treated with Que (25 μM) combined with and without LY294002, the PI3K inhibitor. The results showed that LY294002 suppressed the alleviation effect on Que in H2O2-induced GES-1 cells. In conclusion, the current study demonstrates that Que alleviates oxidative stress damage in GES-1 cells by improving mitochondrial function and mucosal barrier and suppressing inflammation through regulating the PI3K/AKT signaling pathway, indicating the potential therapeutic effects of Que on FD.

Circular RNA circRAB31 acts as a miR-885-5p sponge to suppress gastric cancer progression via the PTEN/PI3K/AKT pathway

Emerging evidence indicated that circular RNAs (circRNAs) play essential roles in cancer progression. A large number of circRNAs have been reported to modulate cancer carcinogenesis. However, the underlying mechanisms by which circRNAs regulate gastric cancer remain largely unclear. By using circRNA microarray, we identified that circRAB31 may serve as a tumor suppressor. circRAB31 was downregulated in gastric cancer tissues and gastric cancer cell lines compared with normal tissues and a human gastric epithelial cell line (GES-1). Overexpression of circRAB31 suppressed gastric cancer proliferation and metastasis in vitro and in vivo, whereas silencing of circRAB31 had the opposite effects. Bioinformatic analysis as well as pull-down and luciferase assays revealed that circRAB31 exerted tumor-suppressive functions by binding directly to miR-885-5p. In addition, we demonstrated that circRAB31 could suppress PI3K/AKT signaling via the phosphatase and tensin homologue (PTEN)—a downstream target gene of miR-885-5p. In summary, our results demonstrated that circRAB31 could serve as a sponge of miR-885-5p to regulate gastric cancer cell proliferation, migration, and invasion by affecting the PTEN/PI3K/AKT signaling.

Long noncoding RNA TUG1 upregulates VEGFA to enhance malignant behaviors in stomach adenocarcinoma by sponging miR-29c-3p.

BackgroundLong noncoding RNA (lncRNA) TUG1 has been reported to display a pivotal role in the tumorigenesis and malignant progression of various types of cancers, including stomach adenocarcinoma (STAD). However, the contribution of aberrant expression of TUG1 and the mechanism by which it serves as a competing endogenous RNA (ceRNA) in STAD remains largely obscure.MethodsThe human STAD cell lines (MGC‐803 and AGS), human normal gastric epithelial cell line (GES‐1), human umbilical vein endothelial cells (HUVECs), and human embryonic kidney cells (HEK293T) were purchased and cultured to investigate the roles of TUG1 in STAD. Twenty BALB/c nude mice were purchased to establish a xenograft model to explore the roles of TUG1 in vivo.ResultsBioinformatics analysis revealed that TUG1 was upregulated in STAD, of which expression was negatively and positively correlated with miR‐29c‐3p and VEGFA, respectively. Functional analyses indicated that TUG1 functioned as an oncogene to promote malignant behaviors (proliferation, migration, and angiogenesis) of STAD cells; whereas miR‐29c‐3p exerted the opposite role. Mechanistically, the interaction between miR‐29c‐3p with TUG1 and VEGFA was demonstrated. It was observed that miR‐29c‐3p could reverse the TUG1‐induced promotion effect on cell proliferation, migration, and angiogenesis in STAD. Furthermore, TUG1 overexpression promoted STAD cell proliferation, metastasis, and angiogenesis, whereas VEGFA silence restored these effects, both in vitro and in vivo.ConclusionThis finding confirmed that lncRNA TUG1 acts as a ceRNA for miR‐29c‐3p to promote tumor progression and angiogenesis by upregulating VEGFA, indicating TUG1 as a therapeutic target in STAD management.