Migration Of Gastric Cancer Cell Lines Research Articles

GGT5 facilitates migration and invasion through the induction of epithelial–mesenchymal transformation in gastric cancer

BackgroundGamma-glutamyltransferase 5 (GGT5), one of the two members in the GGT family (GGT1 and GGT5), plays a crucial role in oxidative regulation, inflammation promotion, and drug metabolism. Particularly in the tumorigenesis of various cancers, its significance has been recognized. Nevertheless, GGT5’s role in gastric cancer (GC) remains ambiguous. This study delves into the function and prognostic significance of GGT5 in GC through a series of in vitro experiments.MethodsEmploying online bioinformatics analysis tools such as The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Kaplan–Meier plotter, and cBioPortal, we explored GGT5 characteristics and functions in GC. This encompassed aberrant expression, prognostic value, genomic alterations and mutations, immune cell infiltration, and associated signaling pathways. Immunohistochemistry was conducted to assess GGT5 expression in GC and adjacent normal tissues. Subsequently, univariate and multivariate logistic regression analyses were applied to investigate the associations between GGT5 and clinical characteristics. CCK8, wound healing, and migration assays were utilized to evaluate the impact of GGT5 on cell viability and migration. Additionally, Gene Set Enrichment Analysis (GSEA) and Western blot analysis were performed to scrutinize the activity of the epithelial–mesenchymal transformation (EMT) signaling pathway under GGT5 regulation.ResultsGGT5 exhibits upregulation in gastric cancer, with its overexpression significantly linked to histological differentiation in GC patients (P < 0.05). Multivariate analysis indicates that elevated GGT5 expression is an independent risk factor associated with poorer overall survival in gastric cancer patients (P < 0.05). In vitro experiments reveal that downregulation of GGT5 hampers the proliferation and migration of GC cell lines. Finally, GSEA using TCGA data highlights a significant correlation between GGT5 expression and genes associated with EMT, a finding further confirmed by Western blot analysis.ConclusionsGGT5 emerges as a promising prognostic biomarker and potential therapeutic target for GC.

Gentiopicroside inhibits the progression of gastric cancer through modulating EGFR/PI3K/AKT signaling pathway

BackgroundThis study was designed to clarify the function and potential mechanism of gentiopicroside (GPS) in regulating the malignant progression of gastric cancer (GC) through in vitro cellular experiments and in vivo animal models.MethodsAGS and HGC27 cells were divided into control group and GPS treatment groups (50 µM and 100 µM). Then, the cellular proliferation, colony formation, migration, invasion, and apoptosis were detected, respectively. Transmission electron microscope (TEM) was used to observe the mitochondrial changes, and the mitochondrial membrane potential (MMP) was determined using the JC-1 commercial kit. Network pharmacology analysis was utilized to screen the potential molecule that may be related to the GPS activity on GC cells, followed by validation tests using Western blot in the presence of specific activator. In addition, xenografted tumor model was established using BALB/c nude mice via subcutaneous injection of HGC27 cells, along with pulmonary metastasis model. Then, the potential effects of GPS on the tumor growth and metastasis were detected by immunohistochemistry (IHC) and HE staining.ResultsGPS inhibited the proliferation, invasion and migration of GC cell lines in a dose-dependent manner. Besides, it could induce mitochondrial apoptosis. Epidermal growth factor receptor (EGFR) may be a potential target for GPS action in GC by network pharmacological analysis. GPS inhibits activation of the EGFR/PI3K/AKT axis by reducing EGFR expression. In vivo experiments indicated that GPS induced significant decrease in tumor volume, and it also inhibited the pulmonary metastasis. For the safety concerns, GPS caused no obvious toxicities to the heart, liver, spleen, lung and kidney tissues. IHC staining confirmed GPS downregulated the activity of EGFR/PI3K/AKT.ConclusionsOur investigation demonstrated for the first time that GPS could inhibit GC malignant progression by targeting the EGFR/PI3K/AKT signaling pathway. This study indicated that GPS may be serve as a safe anti-tumor drug for further treatment of GC.

Second-Generation JK-206 Targets the Oncogenic Signal Mediator RHOA in Gastric Cancer.

Simple SummaryRas homologous A (RHOA), a signal mediator and a GTPase, is associated with the progression of gastric cancer (GC). We present novel RHOA inhibitors designed for greater anti-GC potency by means of lead optimization. The RHOA → BIRC5 signaling circuit was found to be a new therapeutic strategy for regulating GC proliferation and migration.Ras homologous A (RHOA), a signal mediator and a GTPase, is known to be associated with the progression of gastric cancer (GC), which is the fourth most common cause of death in the world. Previously, we designed pharmacologically optimized inhibitors against RHOA, including JK-136 and JK-139. Based on this previous work, we performed lead optimization and designed novel RHOA inhibitors for greater anti-GC potency. Two of these compounds, JK-206 and JK-312, could successfully inhibit the viability and migration of GC cell lines. Furthermore, using transcriptomic analysis of GC cells treated with JK-206, we revealed that the inhibition of RHOA might be associated with the inhibition of the mitogenic pathway. Therefore, JK-206 treatment for RHOA inhibition may be a new therapeutic strategy for regulating GC proliferation and migration.

Bufalin inhibits peritoneal dissemination of gastric cancer through endothelial nitric oxide synthase-mitogen-activated protein kinases signaling pathway.

Peritoneal dissemination threatens the survival of patients with gastric cancer (GC). Bufalin is an extract of traditional Chinese medicine, which has been proved to have anticancer effect. The target of bufalin in suppressing gastric cancer peritoneal dissemination (GCPD) and the underlying mechanism are still unclear. In this research, GC cell line MGC-803 and high-potential peritoneal dissemination cell line MKN-45P were treated with bufalin or L-NAME. Malignant biological behavior and protein level of GC cell lines were detected with MTT, wound healing, transwell, adhesion, and western blotting. Bioinformatics analysis and patient tissues were used to verify the role of endothelial nitric oxide synthase (NOS3) in GC. Mice model was used to assess the effect of bufalin and role of NOS3 in vivo. We found that bufalin inhibited the proliferation, invasion, and migration in GC cell lines. NOS3, which was an independent prognostic factor of GC patients, was predicted to be a potential target of bufalin. Further experiments proved that bufalin reduced the phosphorylation of NOS3, thereby inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway, and ultimately suppressed GCPD by inhibiting EMT process. In conclusion, NOS3 was a potential therapeutic target and prognostic biomarker of GC. Bufalin could suppress GCPD through NOS3-MAPK signaling pathway, which provided more evidence support for intraperitoneal perfusion of bufalin to treat GCPD.

TAFA5 promotes proliferation and migration in gastric cancer.

TAFA chemokine like family member 5 (TAFA5), a TAFA family member that encodes small secreted proteins in the central nervous system, has been demonstrated to have increased expression in human malignancies. However, the expression and function of TAFA5 in gastric cancer (GC) remains unclear. In the present study, public datasets and human GC samples were used to determine the TAFA5 expression levels. The results revealed that TAFA5 was upregulated in GC when compared with adjacent normal tissues. Overexpression of TAFA5 in GC was associated with poor differentiation, and worse tumor, nodal and metastasis stages. In addition, high TAFA5 expression was correlated with unfavorable patient prognoses. In vitro experiments indicated that downregulation of TAFA5 inhibited the proliferation and migration of GC cell lines. Finally, the results from gene set enrichment analysis using data from The Cancer Genome Atlas revealed that TAFA5 expression was significantly correlated with genes associated with epithelial-mesenchymal transition, which was further confirmed by western blot analysis. In conclusion, the results of the present study suggested that TAFA5 had significant effects on GC progression, suggesting that it may serve as a potential therapeutic target for GC therapy.

Interleukin 1 Up-regulates MicroRNA 135b to Promote Inflammation-Associated Gastric Carcinogenesis in Mice

Gastritis is associated with development of stomach cancer, but little is known about changes in microRNA expression patterns during gastric inflammation. Specific changes in gene expression in epithelial cells are difficult to monitor because of the heterogeneity of the tissue. We investigated epithelial cell-specific changes in microRNA expression during gastric inflammation and gastritis-associated carcinogenesis in mice. We used laser microdissection to enrich epithelial cells from K19-C2mE transgenic mice, which spontaneously develop gastritis-associated hyperplasia, and Gan mice, which express activated prostaglandin E2 and Wnt in the gastric mucosa and develop gastric tumors. We measured expression of epithelial cell-enriched microRNAs and used bioinformatics analyses to integrate data from different systems to identify inflammation-associated microRNAs. We validated our findings in gastric tissues from mice and evaluated protein functions in gastric cell lines (SNU-719, SNU-601, SNU-638, AGS, and GIF-14) and knockout mice. Organoids were cultured from gastric corpus tissues of wild-type and miR-135b-knockout C57BL/6 mice. We measured levels of microRNAs in pairs of gastric tumors and nontumor mucosa from 28 patients in Japan. We found microRNA 135b (miR-135B) to be the most overexpressed microRNA in gastric tissues from K19-C2mE and Gan mice: levels increased during the early stages of gastritis-associated carcinogenesis. Levels of miR-135B were also increased in gastric tumor tissues from gp130F/F mice and patients compared with nontumor tissues. In gastric organoids and immortalized cell lines, expression of miR-135B was induced by interleukin 1 signaling. K19-C2mE mice with disruption of Mir-135b developed hyperplastic lesions that were 50% smaller than mice without Mir-135b disruption and had significant reductions in cell proliferation. Expression of miR-135B in gastric cancer cell lines increased their colony formation, migration, and sphere formation. We identified FOXN3 and RECK messenger RNAs (mRNAs) as targets of miR-135B; their knockdown reduced migration ofgastric cancer cell lines. Levels of FOXN3 and RECK mRNAscorrelated inversely with levels of miR-135B in human gastric tumors and in inflamed mucosa from K19-C2mE mice. We found expression of miR-135B to be up-regulated by interleukin L1 signaling in gastric cancer cells and organoids. miR-135B promotes invasiveness and stem-cell features of gastric cancer cells in culture by reducing FOXN3 and RECK messenger RNAs. Levels of these messenger RNA targets, which encode tumor suppressor, are reduced in human gastric tumors.

Long noncoding RNA HOTAIRM1 inhibits cell progression by regulating miR-17-5p/ PTEN axis in gastric cancer.

This study was conducted to identify the significantly altered long noncoding RNAs (lncRNAs), messenger RNA (mRNA) and pathways in gastric cancer (GC). We used microarray analysis to identify differentially expressed lncRNAs and mRNAs, whereas the obviously changed pathways were found by gene set enrichment analysis. The coexpression network of lncRNA and mRNA was constructed by Cytoscape, and their target relationships with miRNAs were predicted by miRcode and TargetScan. qRT-PCR and Western blot were performed to determine the expression levels of mRNAs and proteins in tissues and cell lines. Dual-luciferase reporter assay was applied to achieve the determination of the specific target relationships. Cell viability, migration, and apoptosis were detected by MTT assay, wound healing assay and flow cytometry, respectively. Through the xenograft assay, the gastric tumor was implanted into nude mice to investigate the influence of HOTAIRM1 in vivo. HOTAIRM1 and phosphatase and tensin homolog (PTEN) were both downregulated in GC, whereas miR-17-5p was upregulated. Moreover, the PI3K/AKT pathway was found activated in GC. HOTAIRM1 targeted miR-17-5p, whereas PTEN was the downstream target gene of miR-17-5p. HOTAIRM1 suppressed proliferation and migration of GC cell line and induced their apoptosis, whereas miR-17-5p played the opposite role on GC cell line. HOTAIRM1 also postponed tumor growth in vivo and inhibited the PI3K/AKT pathway in GC. LncRNA HORAIRM1 suppressed the PI3K/AKT pathway in GC and inhibited the progression of GC by serving as a competing endogenous RNA of miR-17-5p, mediating the expression of PTEN.

Fibroblast Activation Protein-α-Positive Fibroblasts Promote Gastric Cancer Progression and Resistance to Immune Checkpoint Blockade.

Gastric cancer (GC) is one of the main causes of cancer death. The tumor microenvironment has a profound effect on inducing tumor growth, metastasis, and immunosuppression. Fibroblast activation protein-α (FAP) is a protein that is usually expressed in fibroblasts, such as cancer-associated fibroblasts, which are major components of the tumor microenvironment. However, the role of FAP in GC progression and treatment is still unknown. In this study, we explored these problems based on GC patient samples and experimental models. We found that high FAP expression was an independent prognosticator of poor survival in GC patients. FAP+ cancer-associated fibroblasts (CAFs) promoted the survival, proliferation, and migration of GC cell lines in vitro. Moreover, they also induced drug resistance of the GC cell lines and inhibited the antitumor functions of T cells in the GC tumor microenvironment. More importantly, we found that targeting FAP+ CAFs substantially enhanced the antitumor effects of immune checkpoint blockades in GC xenograft models. This evidence highly suggested that FAP is a potential prognosticator of GC patients and a target for synergizing with other treatments, especially immune checkpoint blockades in GC.

MicroRNA-497 acts as a tumor suppressor in gastric cancer and is downregulated by DNA methylation.

Gastric cancer (GC) is one of the most common malignant tumors in the world and microRNAs (miRNAs) play an important role in GC. In this study, we found miR‑497 played an important role and served as a novel biomarker in GC. Quantitative real-time PCR (qRT-PCR) was used to measure the miR‑497 expression in GC cell lines and 86paired GC samples and we also analyzed its correlation with GC clinicopathological parameters. A series of cellular function experiments were applied to validate the effects of miR‑497 on GC. In addition, methylation-specific PCR (MSP) was applied to detect the gene methylation status. Finally, the correlation between miR‑497 and the target gene was analyzed by western blotting assay. miR‑497 was reduced obviously in GC cells and tissues and significantly associated with the pathologic stage. Low expression of miR‑497 significantly inhibited the proliferation, invasion and migration of GC cell lines and accelerated apoptosis. Moreover, we found that the aberrant expression of miR‑497 may be ascribed to DNA methylation. microRNA.org and luciferase reporter assay suggested that RAF1 was a direct target of miR‑497 in GC. This study suggested that miR‑497 could serve as a tumor suppressor and a potential early diagnostic marker of GC by targeting Raf-1 proto-oncogene, serine/threonine kinase (RAF1).

Abnormal expression of FOSB correlates with tumor progression and poor survival in patients with gastric cancer.

FOSB protein is encoded by the FOSB gene in humans, which shares structural similarities with the prototype of the Fos family. FOSB plays a role by AP-1 complex which is composed of heterodimers of Jun and Fos members. Our experiment aimed to evaluate the effect of FOSB in gastric cancer (GC) patients and then probe its significance in prognosis. We detected the expression of FOSB in GC and adjacent non-cancerous tissues by western blot analysis and real-time quantitative PCR (qRT-PCR). Moreover, we analyzed FOSB expression in patients who underwent resection procedures using immunohistochemistry. The relationship between the expression of FOSB, the clinicopathological characteristics and the patients survival were also investigated. Furthermore, in vitro, we evaluated the effects of FOSB gene on gastric cancer cell viability, proliferation and migration by MTT, clone formation and transwell assays. Finally, the Kaplan-Meier method and log-rank test were used to compare the overall survival between high FOSB expression group and low FOSB expression group. Immunohistochemical staining data showed that FOSB expression was significantly decreased in gastric cancer cases. In addition, we confirmed FOSB downregulation in both mRNA and protein levels in GC tissues compared with matched adjacent non-cancerous tissues. Downregulated expression of FOSB was correlated with poor differentiation, lymph node metastasis and advanced TNM stage. Moreover, we found that low FOSB expression exhibited a significant correlation with poor prognosis for GC patients by Kaplan-Meier survival analysis. Overexpression of FOSB significantly suppressed cell proliferation, clone formation and migration in GC cell lines. In contrast, silencing of FOSB expression in GC cells promoted proliferation, clone formation and migration. Our results showed that FOSB plays a crucial role in the suppression of GC, and that it may be a useful biomarker in diagnosis and prognosis for GC patients.

Epigenetic suppression of the immunoregulator MZB1 is associated with the malignant phenotype of gastric cancer.

Prediction of tumor recurrence after curative resection is critical for determining the prognosis of patients with gastric cancer (GC). The initiation and progression of GC are associated with inappropriate immune responses caused by chronic inflammation of the gastric mucosa. To identify immunoregulatory molecules involved in GC progression, GC cell lines and 200 pairs of tumor and normal tissues from patients with GC were analyzed for gene expression, amplification and methylation as well as function of a differentially expressed gene. The transcriptome analysis revealed that marginal zone B and B1 cell specific protein (MZB1) was expressed at significantly decreased levels in primary GC tissues when compared with the corresponding normal gastric mucosa. PCR array analysis exploring genes expressed cooperatively with MZB1 revealed that differential expression of MZB1 mRNA in GC cell lines correlated positively with the levels of the mRNAs encoding estrogen receptor 1 and desumoylating isopeptidase 1. Hypermethylation of the MZB1 promoter was frequent in cell lines with decreased levels of MZB1 mRNA. siRNA-mediated knockdown of MZB1 significantly increased proliferation, invasion and migration of GC cell lines. Low MZB1 expression was an independent prognostic factor for recurrence after curative gastrectomy and was associated significantly with increased hematogenous recurrence. MZB1 acts as a suppressor of GC. Low MZB1 expression in the primary GC tissue is predictive of recurrence after curative resection.

Constitutive expression of PPARγ inhibits proliferation and migration of gastric cancer cells and down-regulates Wnt/β-Catenin signaling pathway downstream target genes TERT and ENAH

BackgroundPeroxisome proliferator-activated receptor γ (PPARγ) is a member of the PPAR nuclear hormone receptor superfamily, which plays a crucial role in carcinogenesis. Wnt/β-Catenin signaling pathway has been well certified to contribute to the progression of gastric malignancies. β-Catenin mediates transcriptional regulation by forming a complex with LEF/TCF transcription factors, resulting in activation of downstream target genes such as TERT, ENAH. In this study, we aimed at detecting the effect of PPARγ on TERT, ENAH and explaining the further mechanisms of PPARγ on tumor suppression. MethodsThe pEGFP-N1-PPARγ recombinant plasmid has already been constructed by researchers in our laboratory. We stably transfected it into three gastric cancer (GC) cell lines (MKN-28, SGC-7901 and BGC-823). CCK-8 and transwell assay were employed to analyze the capability of cell proliferation and metastasis. The mRNA and protein levels were evaluated by real-time PCR and western blot analysis. ResultsAfter transfected with PPARγ overexpression plasmid, the ability of cell proliferation and migration declined significantly (p<0.05). The expression of PPARγ increased (p<0.05) and β-Catenin was inhibited obviously (p<0.05) in the group of pEGFP-N1-PPARγ plasmid transfection. Meanwhile, the mRNA or protein levels of TERT and ENAH were suppressed (p<0.05) in pEGFP-N1-PPARγ plasmid transfection group compared with control groups. ConclusionPPARγ might inhibit the proliferation and migration of GC cell lines through suppressing the expression of TERT and ENAH. PPARγ played an important role as a physiological regulator and might be a target for the treatment of GC.

MiR-1303 Targets Claudin-18 Gene to Modulate Proliferation and Invasion of Gastric Cancer Cells

MicroRNAs have emerged as important gene regulators and are recognized as important molecules in carcinogenesis. However, the effects of microRNA-1303 (miR-1303) on gastric cancer (GC) cells and the upstream regulation of GC-associated claudin-18 gene (CLDN18) remain unclear. miR-1303 may be involved in the tumorigenesis of GC by targeting CLDN18. The purpose of this study was to explore the effect of miR-1303 targeting of CLDN18 on the proliferation, migration and invasion of human GC cells. The expression of miR-1303 and claudin-18 in GC tissues and gastric cancer cell lines were detected by qRT-PCR and western blotting, respectively. CCK8 and colony formation assays were performed to study the influence of miR-1303 on the proliferation of the GC cell lines. Transwell and wound-healing assays were carried out to investigate the effect of miR-1303 on the invasion and migration of GC cell lines. Luciferase reporter assays, restore assays and western blotting were used to demonstrate whether CLDN18 is a direct target of miR-1303. miR-1303 was significantly overexpressed whereas claudin-18 was downregulated in GC tissues and cell lines, which was significantly associated with tumor size, location invasion, histologic type and tumor-node-metastasis stage. Cell proliferation rates were reduced, and cell invasion and migratory ability was significantly restricted in miR-1303 inhibitor-transfected groups. miR-1303 could bind to the putative binding sites in CLDN18 mRNA 3'-UTR and visibly lower the expression of claudin-18. The introduction of claudin-18 without 3'-UTR restored the miR-1303 promoting migration function. Downregulation of miR-1303 can inhibit proliferation, migration and invasion of GC cells by targeting CLDN18.