Normal Human Gastric Epithelial Cells Research Articles

Chemical constituents from a soil fungus Xylaria sp. Y01 and their anticancer potential

Fifteen compounds, namely three new cytochalasans, one new sesquiterpenoid, two new amides, eight known cytochalasans, and one known amide, were isolated from the rice medium fermented by a soil fungus Xylaria sp. Y01. The structures of these compounds were determined through comprehensive analyses of their extensive spectroscopic data, along with quantum chemical calculations. In vitro cytotoxicity assays, we selected three cancer cell lines (HCT116, 4T1, and MHCC97H) and one normal human gastric epithelial GES-1 cell line to explore their potential as anticancer agents. As a result, 6,7-dihydro-7-oxo-cytochalasin C, 5,6-dihydro-7-oxo-19,20-epoxycytochalasin C, 19,20-epoxycytochalasin D, and 19‑epi-cytochalasin P1 showed cytotoxicity against GES-1, while only 19,20-epoxycytochalasin D also exhibited cytotoxicity against 4T1. Our findings further enrich the structural diversity of secondary metabolites from Xylaria species and provide clues for exploring their anticancer potential.

Identifying the signature of NAD+ metabolism-related genes for immunotherapy of gastric cancer

NAD (Nicotinamide Adenine Dinucleotide) -related metabolic reprogramming in tumor cells involves multiple vital cellular processes. However, the role of NAD metabolism in immunity and the prognosis of gastric cancer (GC) remains not elucidated. Here we identified and clustered 33 NAD + metabolism-related genes (NMRGs) based on 808 GC samples from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Survival analysis between different groups found a poor prognosis in the GC patients with high NMRGs expression. Gene SGCE, APOD, and PPP1R14A were identified and performed high expression in GC samples, while the qRT-PCR results further confirmed that their expression levels in GC cell lines were significantly higher than those from normal human gastric mucosa epithelial cells. Based on the single-cell analysis, Gene SGCE, APOD, and PPP1R14A can potentially be novel biomarkers of tumor-associated fibroblasts (CAFs). In parallel, the proliferation and migration of GC cells were significantly hampered following the knockdown of SGCE, APOD, and PPP1R14A, particularly APOD, we confirmed that APOD knockdown can inhibit β-catenin and N-cadherin expression, while promote E-cadherin expression. This study unveils a novel NMRGs-related gene signature, highlighting APOD as a prognostic biomarker linked to the tumor microenvironment. APOD drives GC cell proliferation and metastasis through the Wnt/β-catenin/EMT signaling pathway, establishing it as a promising therapeutic target for GC patients.

The Role of MiR-375 in Migration and Invasion of H.pylori-induced Gastric Cancer Cell Model.

This article aimed to investigate the mechanism of miR-375 in Hp-induced gastric cancer cells (GCCs) model. Human normal gastric mucosal epithelial cell (GMEC) line GES-1 and human GCCs strain MKN45 were used as research objects. The expression of miR-375 was detected after H.pylori (Hp) infection of GCCs. The cell activity was detected by, 53-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, and the cell multiplication was determined by cell counting kit-8 (CCK-8) method. Transwell assay was used to detect the effect of cell invasion and migration ability. The expression levels of JAK1 and STAT3 proteins were determined by Western blot (WB). MiR-375 was increased in GCCs after Hp infection, and JAK1, STAT3, p-JAK1, and p-STAT3 in GCCs after Hp infection were visibly increased. In addition, the overexpressed miR-375 promoted the multiplication activity, migration, and invasion ability of GCCs. MiR-375 promotes Hp-induced migration and invasion of GCCs by targeting JAK1/STAT3. This article reveals the important role of miR-375 in Hp-induced GC, which provides new clues for further study of its mechanism and therapeutic targets.

The Cytoprotective Effect of C60 Derivatives in the Self-Microemulsifying Drug Delivery System against Triptolide-Induced Cytotoxicity In Vitro.

The aim of this study was to optimize the formulation of a C60-modified self-microemulsifying drug delivery system loaded with triptolide (C60-SMEDDS/TP) and evaluate the cytoprotective effect of the C60-SMEDDS/TP on normal human cells. The C60-SMEDDS/TP exhibited rapid emulsification, an optimal particle size distribution of 50 ± 0.19 nm (PDI 0.211 ± 0.049), and a near-neutral zeta potential of -1.60 mV. The release kinetics of TP from the C60-SMEDDS/TP exhibited a sustained release profile and followed pseudo-first-order release kinetics. Cellular proliferation and apoptosis analysis indicated that the C60-SMEDDS/TP (with a mass ratio of TP: DSPE-PEG-C60 = 1:10) exhibited lower toxicity towards L02 and GES-1 cells. This was demonstrated by a higher IC50 (40.88 nM on L02 cells and 17.22 nM on GES-1 cells) compared to free TP (21.3 nM and 11.1 nM), and a lower apoptosis rate (20.8% on L02 cells and 26.3% on GES-1 cells, respectively) compared to free TP (50.5% and 47.0%) at a concentration of 50 nM. In comparison to the free TP group, L02 cells and GES-1 cells exposed to the C60-SMEDDS/TP exhibited a significant decrease in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM). On the other hand, the C60-SMEDDS/TP demonstrated a similar inhibitory effect on BEL-7402 cells (IC50 = 28.9 nM) and HepG2 cells (IC50 = 107.6 nM), comparable to that of the free TP (27.2 nM and 90.4 nM). The C60-SMEDDS/TP group also exhibited a similar intracellular level of ROS and mitochondrial membrane potential compared to the SMEDDS/TP and free TP groups. Fullerenol-Grafted Distearoyl Phosphatidylethanolamine-Polyethylene Glycol (DSPE-PEG-C60) was synthesized and applied in the self-microemulsifying drug delivery system. The C60-SMEDDS/TP was formulated using Cremophor EL, medium-chain triglycerides (MCT), PEG-400, and DSPE-PEG-C60, and loaded with triptolide (TP). The toxicity and bioactivity of the C60-SMEDDS/TP were assessed using normal human liver cell lines (L02 cells), normal human gastric mucosal epithelial cell lines (GES-1 cells), and liver cancer cell lines (BEL-7402 cells and HepG2 cells). The production of reactive oxygen species (ROS) after the C60-SMEDDS/TP treatment was assessed using 2',7'-dichlorofluorescein diacetate (DCFDA) staining. The alterations in mitochondrial membrane potential (ΔψM) were assessed by measuring JC-1 fluorescence. The cytoprotection provided by the C60-SMEDDS/TP favored normal cells (L02 and GES-1) over tumor cells (BEL-7402 and HepG2 cells) in vitro. This suggests a promising approach for the safe and effective treatment of TP.

Lonicera japonica Thunb. Ethanol Extract Exerts a Protective Effect on Normal Human Gastric Epithelial Cells by Modulating the Activity of Tumor-Necrosis-Factor-α-Induced Inflammatory Cyclooxygenase 2/Prostaglandin E2 and Matrix Metalloproteinase 9.

Gastric inflammation-related disorders are commonly observed digestive system illnesses characterized by the activation of proinflammatory cytokines, particularly tumor necrosis factor-α (TNF-α). This results in the induction of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PEG2) and matrix metallopeptidase-9 (MMP-9). These factors contribute to the pathogenesis of gastric inflammation disorders. We examined the preventive effects of Lonicera japonica Thunb. ethanol extract (Lj-EtOH) on gastric inflammation induced by TNF-α in normal human gastric mucosa epithelial cells (GES-1). The GES-1 cell line was used to establish a model that simulated the overexpression of COX-2/PGE2 and MMP-9 proteins induced by TNF-α to examine the anti-inflammatory properties of Lj extracts. The results indicated that Lj-EtOH exhibits significant inhibitory effects on COX-2/PEG2 and MMP-9 activity, attenuates cell migration, and provides protection against TNF-α-induced gastric inflammation. The protective effects of Lj-EtOH are associated with the modulation of COX-2/PEG2 and MMP-9 through the activation of TNFR-ERK 1/2 signaling pathways as well as the involvement of c-Fos and nuclear factor kappa B (NF-κB) signaling pathways. Based on our findings, Lj-EtOH exhibits a preventive effect on human gastric epithelial cells. Consequently, it may represent a novel treatment for the management of gastric inflammation.

Astragaloside IV prevents enterovirus 71-induced pyroptosis through the TCF12-TXNIP-Keap1/Nrf2 axis

This study aimed to investigate the inhibitory effect of astragaloside IV (AS-IV) on enterovirus 71 (EV71)-induced pyroptosis. The results indicated that AST-IV inhibited EV71-induced pyroptosis in normal human gastric epithelial (GES-1) cells. Therefore, we investigated the mechanisms underlying pyroptosis inhibition by AST-IV. We found that EV71 inhibited the Nrf2-Keap1 pathway, whereas AST-IV treatment activated it. Overexpression of Nrf2 led to the inhibition of EV71-induced pyroptosis. EV71 infection and AST-IV treatment upregulated and downregulated thioredoxin interacting protein (TXNIP), respectively. Downregulation of TXNIP activated the Keap1/Nrf2 signaling pathway and inhibited EV71-induced pyroptosis. Furthermore, transcription factor 12 (TCF12) reduced the protein and mRNA levels of TXNIP, and AST-IV treatment upregulated TCF12. These results demonstrate that the TCF12-TXNIP-Keap1/Nrf2 axis participates in the inhibition of pyroptosis by AST-IV in EV71-infected GES-1 cells, suggesting that AST-IV could be used as a dietary supplement to alleviate EV71-induced pyroptosis.

Microfluidic device featuring micro-constrained channels for multi-parametric assessment of cellular biomechanics and high-precision mechanical phenotyping of gastric cells

BackgroundCellular biomechanics plays a significant role in the regulation of cellular physiological and pathological processes. In recent years, multiple methods have been developed to evaluate cellular biomechanics, such as atomic force microscopy (AFM), micropipette aspiration, and magnetic tweezers. However, most of these methods only focus on a single parameter and cannot automate the process at a high-efficiency level. A novel microfluidic method is necessary to achieve the simultaneous multi-parametric measurement of cellular biomechanics and high-precision cellular mechanical phenotyping at high throughput. ResultsTo tackle the issue concerning the low-throughput and cellular single-parameter evaluation, we designed and fabricated a microfluidic chip featuring multiple micro-constrained channels structure, providing a simultaneous multi-parametric assessment of cellular biomechanics, including elastic modulus, recovery capability, and deformability. We compared the biomechanical properties of normal human gastric mucosal epithelial cells (GES-1) and human gastric cancer cells (AGS and MKN-45) by the chip. Results demonstrated that the elastic modulus of GES-1, AGS, and MKN-45 cells decreased sequentially, which was the opposite of their invasiveness and metastasis potential, suggesting the inverse correlation between cellular elastic modulus and malignancy. Meanwhile, the recovery capability and deformability of GES-1, AGS, and MKN-45 cells increased sequentially, demonstrating the positive correlation between cellular deformability and malignancy. Furthermore, multiple parameters were used to distinguish gastric cancer cells from normal gastric cells via machine learning. An accuracy of over 94.8% for identifying gastric cancer cells was achieved. SignificanceThis study provides a deep insight into the biophysical mechanism of gastric cancer metastasis at the single-cell level and possesses great potential to function as a valuable tool for single-cell analysis, thereby facilitating high-precision and high-throughput discrimination of cellular phenotypes that are not easily discernible through single-marker analysis.

Single-cell data revealed CD14-type and FCGR3A-type macrophages and relevant prognostic factors for predicting immunotherapy and prognosis in stomach adenocarcinoma.

Stomach adenocarcinoma (STAD) exhibits profound tumor heterogeneity and represents a great therapeutic challenge. Single-cell sequencing technology is a powerful tool to identify characteristic cell types. Single-cell sequencing data (scRNA-seq) GSE167297 and bulk RNA-seq data from TCGA, GTEx, GSE26901 and GSE15459 database were included in this study. By downscaling and annotating the cellular data in scRNA-seq, critical cell types in tumor progression were identified by AUCell score. Relevant gene modules were then identified by weighted gene co-expression network analysis (WGCNA). A prognostic scoring system was constructed by identifying prognostic factors in STAD by Least absolute shrinkage and selection operator (LASSO) COX model. The prognosis and model performance in the RiskScore groups were measured by Kaplan-Meier (K-M) curves and Receiver operating characteristic (ROC) curves. Nomogram was drawn based on RiskScore and prognosis-related clinical factors. In addition, we evaluated patient's feedback on immunotherapy in the RiskScore groups by TIMER, ESTIMATE and TIDE analysis. Finally, the expression levels of prognostic factors were verified in gastric cancer cell lines (MKN7 and MKN28) and human normal gastric mucosal epithelial cells (GES-1), and the effects of prognostic factors on the viability of gastric cancer cells were examined by the CCK8 assay and cell cycle. scRNA-seq analysis revealed that 11 cell types were identified, and macrophages exhibited relatively higher AUCell scores and specifically expressed CD14 and FCGR3A. High macrophage scores worsened the prognosis of STAD patients. We intersected the specifically expressed genes in macrophages subgroups (670) and macrophage module genes (2,360) obtained from WGCNA analysis. Among 86 common genes, seven prognostic factors (RGS2, GNAI2, ANXA5, MARCKS, CD36, NRP1 and PDE4A) were identified and composed a RiskScore model. Patients in low Risk group showed a better survival advantage. Nomogram also provided a favorable prediction for survival at 1, 3 and 5 years in STAD patients. Besides, we found positive feedback to immunotherapy in patients with low RiskScore. The expression tendency of the seven prognostic factors in MKN7 and MKN28 was consistent with that in the RNA-seq data in addition to comparison of protein expression levels in the public HPA (The Human Protein Atlas) database. Further functional exploration disclosed that MARCKS was an important prognostic factor in regulating cell viability in STAD. This study preliminary uncovered a single cell atlas for STAD patients, and Macrophages relevant gene signature and nomogram displayed favorable immunotherapy and prognostic prediction ability. Collectively, our work provides a new insight into the molecular mechanisms and therapeutic approach for LUAD patients.

Forkhead Box M1 Regulates the Proliferation,Invasion,and Drug Resistance of Gastric Cancer Cells via circ_NOTCH1

Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.

Effect of neutralization treatment on properties of chitosan/bamboo leaf flavonoids/nano-metal oxide composite films and application of films in antioxidation of rapeseed oil

Neutralization treatment improved the slow-release antioxidant food packaging function of chitosan (CS)/bamboo leaf flavone (BLF)/nano-metal oxides composite films. The film cast from the CS composite solution neutralized by KOH solution had good thermal stability. The elongation at break of the neutralized CS/BLF film was increased by about 5 times, which provided the possibility for its packaging application. After 24 h of soaking in different pH solutions, the unneutralized films swelled severely and even dissolved, while the neutralized films maintained the basic structure with a small degree of swelling, and the release trend of BLF conformed to the logistic function (R2 ≥ 0.9186). The films had a good ability to resist free radicals, which was related to the release amount of BLF and the pH of the solution. The antimicrobial neutralized CS/BLF/nano-ZnO film, like the nano-CuO and Fe3O4 films, were effective in inhibiting the increase in peroxide value and 2-thiobarbituric acid induced by thermal oxygen oxidation of rapeseed oil and had no toxicity to normal human gastric epithelial cells. Therefore, the neutralized CS/BLF/nano-ZnO film is likely to become an active food packaging material for oil-packed food, which can prolong the shelf life of packaged food.

Cancerous inhibitor of protein phosphatase 2A enhances chemoresistance of gastric cancer cells to oxaliplatin.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a newly discovered oncogene. It is an active cell proliferation regulatory factor that inhibits tumor apoptosis in gastric cancer (GC) cells. CIP2A is functionally related to chemoresistance in various types of tumors according to recent studies. The underlying mechanism, however, is unknown. Further, the primary treatment regimen for GC is oxaliplatin-based chemotherapy. Nonetheless, it often fails due to chemoresistance of GC cells to oxaliplatin. The goal of this study was to examine CIP2A expression and its association with oxaliplatin resistance in human GC cells. Immunohistochemistry was used to examine CIP2A expression in GC tissues and adjacent normal tissues. CIP2A expression in GC cell lines was reduced using small interfering RNA. After confirming the silencing efficiency, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium and flow cytometry assays were used to evaluate cell proliferation and apoptosis caused by oxaliplatin treatment. Further, the key genes and protein changes were verified using real-time quantitative reverse transcription PCR and Western blotting, respectively, before and after intervention. For bioinformatics analysis, we used the R software and Bioconductor project. For statistical analysis, we used GraphPad Prism 6.0 and the Statistical Package for the Social Sciences software version 20.0 (IBM, Armonk, United States). A high level of CIP2A expression was associated with tumor size, T stage, lymph node metastasis, Tumor Node Metastasis stage, and a poor prognosis. Further, CIP2A expression was higher in GC cells than in normal human gastric epithelial cells. Using small interfering RNA against CIP2A, we discovered that CIP2A knockdown inhibited cell proliferation and significantly increased GC cell sensitivity to oxaliplatin. Moreover, CIP2A knockdown enhanced oxaliplatin-induced apoptosis in GC cells. Hence, high CIP2A levels in GC may be a factor in chemoresistance to oxaliplatin. In human GC cells, CIP2A regulated protein kinase B phosphorylation, and chemical inhibition of the protein kinase B signaling pathway was significantly associated with increased sensitivity to oxaliplatin. Therefore, the protein kinase B signaling pathway was correlated with CIP2A-enhanced chemoresistance of human GC cells to oxaliplatin. CIP2A expression could be a novel therapeutic strategy for chemoresistance in GC.

Propofol-Induced miR-493-3p Inhibits Growth and Invasion of Gastric Cancer through Suppression of DKK1-Mediated Wnt/β-Catenin Signaling Activation.

Gastric cancer (GC) is one of the most common malignant tumors and also one of the most deadly tumors. In recent years, studies have shown that propofol can inhibit the proliferation and metastasis of many tumor cells. In the present study, we aimed to investigate the underlying mechanism of propofol inhibition of the growth and invasion of GC cells. Human gastric cancer cell line SGC-7901 and human normal gastric epithelial cell GES-1 were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Propofol of different concentrations (0, 2, 5, and 10 μg/mL) was used to treat SGC-7901, and miR-493-3p inhibitor was transfected into SGC-7901. The cell proliferation of SGC-7901 was analyzed by MTT as well as colony formation assay. The qRT-PCR was used to assess the expression of mRNA for key genes. We examined the protein expression of DKK1 and relative markers with western blot. Putative binding places of miR-493-3p on the 3'-untranslated area of DKK1 were predicted using bioinformatics and dual-luciferase method. Propofol prohibited phenotypic features of GC, according to our findings. Furthermore, research into the underlying mechanisms of propofol's suppressive effects in GC cell proved that propofol therapy improved the degrees of expression of the potential tumor suppressor miR-493-3p. The inhibiting properties of propofol on GC cell development, migration, and invasion were abolished when propofol-induced miR493-3p was silenced with anti-miR-493-3p. We also found that this drug reversed epithelial-mesenchymal transformation in SGC-7901 cells via inducing miR-493-3p. Propofol-induced miR-493-3p decreases GC cell development via targeting DKK1 and hence inhibits Wnt/β-catenin signaling, according to these findings. Propofol-induced miR-493-3p decreased GC cell development via targeting DKK1 and hence inhibited Wnt/β-catenin signaling, according to these findings.

FREEZE-DRIED RASPBERRY POMACE: A RICH RESERVOIR OF POLYPHENOLS WITH ANTIOXIDANT AND PROTECTIVE EFFECTS AS A PROMISING FUNCTIONAL FOOD ADDITIVE

Background. Raspberries (Rubus idaeus L.) hold significant importance in the food industry. Rasp berry pomace, commonly regarded as a waste product during fruit processing, is a rich source of biologi cally active components. This study aimed to assess the detailed profile of polyphenols in fresh and freeze dried raspberry pomace. Furthermore, we examined its potential to protect normal human gastric epithelial cells against oxidative stress in an H2O2-induced stress model. A qualitative and quantitative analysis of phenolic compounds was performed using the high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) method. Cell viability was assessed based on the integrity of lysosomal membranes (neutral red NR assay) and mitochondrial dehydrogenase activity (MTT). Additionally, the reducing potential (FRAP), the ability to scavenge free radicals in the DPPH• test and studies on ABC HP021X cells based on the H2DCFDA test were evaluated. Results and conclusion. It was found that the extract from freeze-dried raspberry pomace is a rich source of various polyphenolic compounds, including ellagitannins, epicatechin and anthocyanins. The extract was characterized by high antioxidant potential and exhibited a protective effect on human gastric epithelial cells against H2O2-induced cytotoxicity. Cell incubation with the extracts prior to oxidative stress induction attenuated H2O2-induced cytotoxicity in a concentration-dependent manner, and at a con centration of 250 µg/cm3 , it abolished the detrimental effects of H2O2. Additionally, the extract at this concentration restored the redox status in cells with induced oxidative stress and reduced ROS levels to values similar to the control. Thus, raspberry pomace extracts may be considered a valuable food supplement.

Low Expression of GIGYF1 Inhibits Metastasis, Proliferation, and Promotes Apoptosis and Autophagy of Gastric Cancer Cells.

GRB10 interacting GYF protein 1 (GIGYF1) binds to the N-terminal region of Grb10, regulates multiple signaling pathways. However, it is not clear what happens to cell proliferation, metastasis, apoptosis, and autophagy when the expression level of GIGYF1 gene is reduced. Detection of GIGYF1 expression in clinical tissue specimens and gastric cancer (GC) cell lines by quantitative Real-time PCR (qRT-PCR), GIGYF1 gene was knocked down in MGC-803 cells using small interfering RNA, the effect of GIGYF1 gene on cell metastasis was detected using Transwell assay and wound healing assay, the effect on cell proliferation was detected using plate cloning assay and cck-8 assay, the effect on apoptosis was detected using flow cytometry, autophagosomes were detected using laser confocal microscopy, and the effect on protein expression was detected using immunofluorescence and Western blotting. GIGYF1 gene expression was higher in tumor tissue samples than in paracancer tissue samples, and higher in human GC cell lines than in human normal gastric epithelial cells. GIGYF1 gene knockdown inhibited cell migration, scratch healing ability and EMT process, weakened cell proliferation ability, increased apoptosis rate, promoted the formation of autophagosomes, and changed the corresponding protein expression level. Meanwhile, GIGYF1 knockdowns inhibited the ERK and AKT signaling. In conclusion, the low expression of GIGYF1 gene can inhibit the occurrence and progression of gastric cancer, during which the ERK and AKT signaling pathways are inhibited.

ISLR interacts with MGAT5 to promote the malignant progression of human gastric cancer AGS cells.

Gastric cancer is a common malignant tumor with high morbidity and mortality. The present study aimed to investigate the role of the immunoglobulin superfamily containing leucine-rich repeat (ISLR) gene in gastric cancer and examine whether ISLR could interact with N-acetylglucosaminyltransferase V (MGAT5) to affect the malignant progression of gastric cancer. The expression of ISLR and MGAT5 in human normal gastric epithelial cells and human gastric cancer cells, and the transfection efficiency of ISLR interference plasmids and MGAT5 overexpression plasmids were all detected by reverse transcription-quantitative PCR (RT-qPCR) and western blot. The viability, proliferation, migration and invasion, and epithelial-mesenchymal transition (EMT) of gastric cancer cells after indicated transfection were detected by Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, wound healing assay, and transwell assay. The interaction between ISLR and MGAT5 was confirmed by co-immunoprecipitation. The expression of proteins related to migration, invasion, and EMT was detected by immunofluorescence and western blot. As a result, ISLR was highly expressed in gastric cancer and was associated with poor prognosis. Interference with ISLR inhibited the viability, proliferation, migration, invasion, and EMT of gastric cancer cells. ISLR interacted with MGAT5 in gastric cancer cells. MGAT5 overexpression weakened the effects of ISLR knockdown on suppressing the viability, proliferation, migration, invasion, and EMT of gastric cancer cells. ISLR interacted with MGAT5 to promote the malignant progression of gastric cancer.

PSMB2 knockdown suppressed proteasome activity and cell proliferation, promoted apoptosis, and blocked NRF1 activation in gastric cancer cells.

Proteasome 20S Subunit Beta 2 (PSMB2) has been suggested to play several roles in cancer. However, the role of PSMB2 and its underlying mechanisms in gastric cancer have not been studied. In this study, qRT-PCR was employed to detect the expression of genes that encode for 26s proteasome subunit proteins. PSMB2 expression and its prognostic ability were assessed by collecting patient tissue samples and reviewing the TCGA and Kaplan-Meier Plotter databases. Immunofluorescence and western blotting experiments were performed to evaluate the expression of PSMB2 in human gastric cancer cells and normal gastric epithelial cells. Subsequently, PSMB2 was knocked down in HGC-27 and SNU-1 cells and overexpressed in N-87 and AGS cells. Proteasome activity assays, 5-Ethynyl-2'-deoxyuridine staining, and TUNEL assays were used to assess proteasome activity, cell proliferation, and apoptosis. Tumor xenograft assays were conducted to evaluate PSMB2 function in vivo. Our results showed that a total of 8 genes encoding for the 26s proteasome subunit protein were highly expressed in a variety of gastric cancer cells. Next, PSMB2 was selected as the focus of subsequent studies which showed that PSMB2 was highly expressed in samples of gastric cancer tissue. Furthermore, a review of the TCGA database revealed that a high level of PSMB2 expression was associated with a poor clinical prognosis. Our results indicated that PSMB2 overexpression promoted proteasome activity, cell proliferation, and suppressed the apoptosis of gastric cancer cells, while those effects were reversed by treatment with a proteasome inhibitor (MG132). In contrast, PSMB2 knockdown produced the opposite effects and also blocked NRF1 activation. Moreover, PSMB2 knockdown inhibited tumor growth in vivo, decreased PSMB2 expression and cell proliferation, and promoted apoptosis in tumor tissues. Our findings revealed the role played by PSMB2 in gastric cancer and suggest PSMB2 as a new target molecule for use in diagnosing and treating gastric cancer.

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.

Abstract 5293: Rationally developing antibody drug conjugates targeting genomically stable gastric cancer

Abstract Background: Gastric cancer (GC) ranks the fifth most common cancer and the fourth most frequent cause of cancer-related deaths. Genomically stable gastric cancers (GSGCs) represent 20% of all GCs and GSGC patients have the worst prognosis among all GC subtypes. Although two HER2-targeted antibody-drug conjugates (ADCs), RC48 and DS8201, have shown promising clinical efficacies, most GSGC patients do not benefit from these ADCs due to the infrequent and heterogeneous HER2 expression. Therefore, there remains a significant and unmet medical need for developing novel ADCs targeting GSGC tumors. Methods: In this presented study, a quantitative and unbiased approach was first used to identify potential targets for GSGC in a panel of human GSGC cell lines in reference with two normal human gastric epithelial cells. Cell membrane localizations of potential targets on GSGC cell lines were determined by immunofluorescent (IF) staining. To correlate it with clinically-relevant GSGC tumor samples, immunohistochemical (IHC) staining of potential targets were performed in human diffuse GC tumor tissues along with normal gastric tissues. Next, GSGC cellular internalization capability of antibodies against potential targets was evaluated. Then, an optimized target was selected for developing GSGC-targeted ADCs. A series of ADC formulations with different linkers and payloads were constructed and characterized. Their anti-tumor activities against GSGC cell lines were subsequently screened in order to identify the optimized ADC formulation. Eventually, the anti-tumor activity and biosafety profile of the optimized ADC formulation were determined in suppressing tumor growth, progression and metastasis by using established orthotopic and peritoneal metastasis models of GSGC. Results: We first identified CD54 as a potential molecular target for human GSGCs using comparative flow cytometric analysis. We next evaluated its potential as an ADC target for GSGC-targeted therapy by determining its overexpression levels, cell membrane localizations, and cell internalization activity in GSGC cells. Furthermore, we constructed and characterized a panel of CD54-targeted ADCs and identified an optimized ADC formulation by quantitatively determining their half maximum inhibitory concentrations (IC50s) and validated its anti-tumor efficacy against GSGC tumors. Meanwhile, there was no evidence to indicate histopathological damages to normal organs in the optimized ADC treated group. Conclusions: In this study, we identified a molecular target for GSGC using an unbiased and quantitative screening assay and explored its potential application in developing GSGC-targeted ADCs. In order to maximize its efficacy, we optimized the ADC formulations against GSGC cells using different ADC linkers and payloads. Our study provides a promising targeted therapeutic candidate for the clinical treatment of GSGC. Citation Format: Rui Xu, Li Yuan, Xiao-Qing Guan, Shi-Li Yao, Bing Zhu, Tong Yang, Chun Liu, Peng Guo, Jiang-Jiang Qin, Xiang-Dong Cheng. Rationally developing antibody drug conjugates targeting genomically stable gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5293.

Natural Compound 2-Chloro-1,3-dimethoxy-5-methylbenzene, Isolated from Hericium Erinaceus, Inhibits Fungal Growth by Disrupting Membranes and Triggering Apoptosis.

In this study, 2-chloro-1,3-dimethoxy-5-methylbenzene (CDM), a natural product with anti-Candida albicans activity, was discovered from the Hericium erinaceus mycelium. The minimum inhibitory concentration of CDM was 62.5 μg/mL. Moreover, structural analogues of CDM obtained from chemical synthesis were applied to explore the structure-activity relationship (SAR) of CDM against C. albicans. It was found that methoxy groups, halogen atoms (except fluorine atoms), and methoxy-meta-position methyl groups in the structure of CDM were the key active groups. Furthermore, we investigated the anti-C. albicans mechanism of CDM. Experiments suggested that CDM destroyed the cell membrane of C. albicans, including the cytoplasmic membrane and mitochondrial membrane, and caused the accumulation of reactive oxygen species and mitochondrial dysfunction, which ultimately led to apoptosis of C. albicans. In addition, CDM had no toxicity on human normal gastric mucosal epithelial cells exposed to a concentration of 125 μg/mL. Experiments showed that CDM reduced the damage of C. albicans to the visceral tissue of infected mice and improved the survival rate of mice. Our research provides a scientific basis for the discovery of effective and safe anti-C. albicans drugs from H. erinaceus.

Cancer-suppressing miR-520-3p gene inhibits proliferation, migration, and invasion of gastric cancer cells through targeted regulation of KLF7

Gastric cancer (GC) is among the most common malignant tumors. Numerous studies have reported that microRNAs (miRNAs) play significant roles in carcinogenesis and treatment. An miRNA, miR-520-3p, has been identified as a cancer-suppressing gene in several cancers. However, the role and underlying mechanism of miR-520-3p regulation of GC remain unknown. In this study, the expression levels of miR-520-3p in cancer tissues of patients with GC -and in adjacent normal tissues, gastric cancer cell lines, and human normal gastricepithelial cells- were detected by qRT-PCR. RNA interference was performed in GC cell lines. After the corresponding treatment, the cells were characterized in vitro or in vivo to evaluate their molecular function. CCK-8, cell colony formation, and a Transwell assay were used to detect cell proliferation rate, viability, and invasion ability. A dual-luciferase reporter gene experiment was used to explore the potential molecular mechanisms of miR-520-3p. The results showed that the expression of miR-520-3p was significantly downregulated in GC tissues and cells, and upregulation of miR-520-3p could inhibit the proliferation, vitality, and invasion of GC cells both in vivo and in vitro. The expression of Kruppel-like factor 7 (KLF7) was greatly upregulated in GC tissues. MiR-520-3p can adsorbKLF7 in GC cells, and KLF7 can reverse the inhibitory effect of miR-520-3p overexpression on the proliferation of GC cells. This study revealed that miR-520-3p plays a significant role in inhibiting the proliferation, invasion, and migration of GC cells by targeting KLF7. These data demonstrate that miR-520-3p may serve as a novel prognostic biomarker and a potential therapeutic target for GC.