Sulfasalazine,a potent suppressor of gastric cancer proliferation and metastasis by inhibition of xCT: Conventional drug in new use

The aim of this study was to explore the role of sulfasalazine on proliferation and metastasis in gastric cancer by inhibition of xCT. The relationships between clinical characteristics and xCT expression were analysed. An immunohistochemical staining assay and Western blot were performed among gastric cancers and normal gastric tissues. qPCR and Western blot were also used to evaluate the mRNA and protein expression in the normal gastric cell and eight gastric cancer cells, respectively. CCK-8 and colony formation assays were used to evaluate the effect of sulfasalazine on the proliferation and colony formation ability of three gastric cancers. The effect of sulfasalazine on the migration and invasion abilities of three cancer cells was assessed by the Transwell assay. xCT protein is up-regulated in gastric cancer specimens and cells. Three gastric cancer cells with high, medium and low expression of xCT were selected for the following analyses. CCK-8 assays revealed that sulfasalazine could attenuate the proliferation of HGC-27 and AGS. Also, the colony formation assay revealed that sulfasalazine might attenuate the colony formation ability in HGC-27 and AGS cells. Plus, the Transwell assays demonstrated that sulfasalazine might attenuate the migration and invasion abilities in HGC-27 and AGS cells. In conclusion, higher expression of xCT is associated with advanced tumour stage and poor overall survival of gastric cancer. Sulfasalazine can attenuate the proliferation, colony formation, metastasis and invasion of gastric cancer in vitro. Further study is required to validate our findings.     

三黄泻心汤对胃癌细胞增殖的影响

目的:探讨三黄泻心汤对胃癌细胞增殖的影响和机制。方法:以体外培养的胃癌细胞系HGC-27为研究对象,给予三黄泻心汤处理后,采用流式细胞仪检测三黄泻心汤对胃癌细胞系HGC-27细胞周期的影响,免疫荧光检测HGC-27细胞Ki67的表达,定量PCR实验检测增殖相关非编码RNA H19表达的影响。结果:当没有幽门螺旋杆菌时,三黄泻心汤对胃癌细胞系的细胞周期、Ki67及lnc RNA H19的表达均无显著影响;而当存在一定浓度的幽门螺旋杆菌时,三黄泻心汤时处于分裂间期和前期(G0/G1期)的细胞的百分比显著的升高,处于DNA复制(S期)的细胞比例及Ki67、lnc RNA H19的表达均明显的降低(P0.05)。结论:在幽门螺旋杆菌存在时,三黄泻心汤显著抑制胃癌细胞系的增殖,可能与降低长链非编码RNA H19的表达有关。     

Urokinase plasminogen activator receptor is upregulated by Helicobacter pylori in human gastric cancer AGS cells via ERK, JNK, and AP-1

The gastric pathogen Helicobacter pylori (H. pylori) is suggested to be associated with gastric cancer progression. In this study, we investigated the effect of H. pylori on urokinase plasminogen activator receptor (uPAR) expression which has been known to correlate closely with gastric cancer invasion. H. pylori induced the uPAR expression in a time- and concentration-dependent manner. Specific inhibitors and inactive mutants of MEK-1 and JNK were found to suppress the H. pylori-induced uPAR expression and the uPAR promoter activity. Electrophoretic mobility shift assay and transient transfection study using an AP-1 decoy oligonucleotide confirmed that the activation of AP-1 is involved in the H. pylori-induced uPAR upregulation. The AGS cells treated with H. pylori showed a remarkably enhanced invasiveness, and this effect was partially abrogated by uPAR-neutralizing antibodies. These results suggest that H. pylori induces uPAR expression via Erk-1/2, JNK, and AP-1 signaling pathways and, in turn, stimulates the cell invasiveness in human gastric cancer AGS cells.     

Ligation or cross-linking of CD40 has different effects on AGS gastric cancer cells

A gastric cancer (GC) cell line, AGS, has high-level expression of CD40, a tumor necrosis factor receptor (TNFR) family member. CD40 is present on the surfaces of a large variety of cells, including B cells, endothelial cells, dendritic cells and some carcinoma cells, and delivers signals regulating diverse cellular responses, such as proliferation, differentiation, growth suppression, and cell death. In this research, we studied the effects of different forms of CD40 stimulation on AGS cells by flow cytometry, Western blotting and siRNA transfection. We found that different forms of CD40 stimulation, either recombinant soluble CD40L (sCD40L, ligation) or agonist anti-CD40 antibody (cross-linking), induced different effects in AGS gastric cancer cells, proliferation or apoptosis. We also showed that VEGF provided a significant contribution to sCD40L-induced proliferation, while agonist anti-CD40 antibody induced GADD45 upregulation and promoted apoptosis.     

Inhibition of human gastric H(+)-K(+)-ATPase alpha-subunit gene expression by Helicobacter pylori

Clinical studies and in vitro data from isolated parietal cells suggest that acute Helicobacter pylori infection inhibits acid secretion. Gastric acidification is mediated by H(+)-K(+)-ATPase, an integral protein of parietal cell apical membranes. To test the hypothesis that H. pylori downregulates H(+)-K(+)-ATPase alpha-subunit (HKalpha) gene expression and to identify potential intracellular signaling pathways mediating such regulation, we transfected human gastric adenocarcinoma (AGS) cells with human and rat HKalpha 5'-flanking DNA fused to a luciferase reporter plasmid. Histamine caused dose-dependent, cimetidine-sensitive (10(-4) M) increases in cAMP, free intracellular Ca(2+), and HKalpha promoter activation in AGS cells. H. pylori infection of transfected AGS cells dose dependently inhibited basal and histamine-stimulated HKalpha promoter activity by 80% and 66%, respectively. H. pylori dose dependently inhibited phorbol myristate acetate-induced (10(-7) M) and staurosporine- (10(-7) M) and calphostin C-sensitive (5 x 10(-8) M) activation of HKalpha promoter. Also, H. pylori inhibited epidermal growth factor (EGF) (10(-8) M), genistein-sensitive (5 x 10(-5) M) activation of HKalpha promoter, reducing activity to 60% of basal level. These data suggest that H. pylori inhibits HKalpha gene expression via intracellular pathways involving protein kinases A and C and protein tyrosine kinase, AGS cells have functional histamine H(2) and EGF receptors, and transiently transfected AGS cells are a useful model for studying regulation of HKalpha gene expression.     

Upregulation of progranulin by Helicobacter pylori in human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway: role in epithelial cell proliferation and migration

Helicobacter pylori is a major human pathogen associated with gastric diseases such as chronic active gastritis, peptic ulcer, and gastric carcinoma. The growth factor progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. We aimed to determine the molecular mechanisms by which H. pylori upregulates the expression of PGRN and the relationship between H. pylori infection and production of PGRN in controlling cell proliferation and migration. Levels of PGRN were examined in gastric tissues from patients and in vitro in gastric epithelial cells. Cell proliferation was measured by colony formation assay. Cell migration was monitored by wound healing migration assay. PGRN protein levels were increased in patients with gastritis and gastric cancer tissue. Infection of gastric epithelial cells with H. pylori significantly increased PGRN expression in a time-dependent manner. Blockade of the p38 and MEK1/2 pathway by inhibitor inhibited H. pylori-mediated PGRN upregulation. Activation of p38 and MEK1/2 pathway by H. pylori was also identified. Knockdown of PGRN attenuated the H. pylori-induced proliferative activity and migration of cancer cells. These findings suggest that the upregulation of PGRN in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.     

LINC00665 induces gastric cancer progression through activating Wnt signaling pathway

Long noncoding RNAs (lncRNAs) are recently recognized as noteworthy regulators of different tumors, counting gastric cancer (GC). Lately, long intergenic noncoding RNA (LINC) 00665 has been verified to display significant parts in several cancers. Be that as it may, its role and mechanism in GC movement still stay uninvestigated. As of now, we observed LINC00665 was obviously GC cells (MKN28, BGC-823, SGC7-901, AGS, HGC-27) in comparison to GES-1 cells, which was identified as human normal gastric epithelial cells. Then, LINC00665 was obviously downregulated in GC cells including AGS and BGC-823 cells. Loss of LINC00665 greatly repressed AGS and BGC-823 cell survival and cell expansion. Moreover, GC cell apoptosis was significantly induced by the loss of LINC00665. For another, we found that the GC cell cycle was also captured in G1 and G2 phases. The experiments on cell migration and invasion indicated that knockdown of LINC00665 restrained GC cell migration and invasion. Modifications in Wnt signaling are closely associated with the development of cancers. Here, we found that Wnt signaling was significantly inactivated by the silence of LINC00665 in GC cells. β-catenin and cyclinD1 were restrained whereas GSK-3β was induced by the inhibition of LINC00665 in GC cells. Furthermore, we confirmed the impact of LINC00665 in vivo using xenograft models. Taken these together, we indicated that LINC00665 could function as a novel biomarker in GC progression.     

下调lncRNA MCF2L-AS1靶向miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡

摘要 目的：探讨lncRNA MCF2L-AS1对胃癌细胞恶性生物学行为的影响及分子机制。方法：选取45例胃癌患者的癌组织及癌旁正常组织,或培养胃黏膜上皮细胞GES-1、胃癌细胞HGC-27,采用RT-qPCR检测MCF2L-AS1和miR-33b-5p的表达水平。采用双荧光素酶报告实验检测MCF2L-AS1和miR-33b-5p的靶向关系。将HGC-27细胞分为si-NC组、si-MCF2L-AS1组、mimic NC组、miR-33b-5p mimic组、si-MCF2L-AS1+inhibitor NC组、si-MCF2L-AS1+miR-33b-5p inhibitor组,分别转染si-NC、si-MCF2L-AS1、mimic NC、miR-33b-5p mimic或共转染si-MCF2L-AS1+inhibitor NC、si-MCF2L-AS1+miR-33b-5p inhibitor。采用MTT实验检测细胞增殖情况,流式细胞术检测细胞凋亡率,克隆形成实验检测细胞克隆形成数,Transwell实验检测迁移和侵袭细胞数。结果：与癌旁正常组织或GES-1细胞相比,胃癌组织或HGC-27细胞中MCF2L-AS1表达水平升高、miR-33b-5p表达水平降低,差异均有统计学意义（P<0.05）。MCF2L-AS1可靶向调控miR-33b-5p。下调MCF2L-AS1或过表达miR-33b-5p,miR-33b-5p表达水平升高,HGC-27细胞凋亡率升高,但细胞增殖、克隆形成数、迁移和侵袭数均减少,差异均有统计学意义（P<0.05）。抑制miR-33b-5p可减弱下调MCF2L-AS1对HGC-27细胞的生物学作用。结论：下调MCF2L-AS1通过上调miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡;MCF2L-AS1通过靶向调控miR-33b-5p表达进而参与胃癌细胞的恶性生物学行为。     

Demonstration and characterization of mutations induced by Helicobacter pylori organisms in gastric epithelial cells

BACKGROUND: Helicobacter pylori gastritis increases gastric cancer risk. Microsatellite instability-type mutations are secondary to deficient DNA mismatch repair. H. pylori gastritis is more frequent in patients with microsatellite instability-positive gastric cancers, and H. pylori organisms independently of inflammation can reduce DNA mismatch repair protein levels, raising the hypothesis that H. pylori organisms might lead to mutagenesis during infection. MATERIALS AND METHODS: Mutations were detected using a green fluorescent protein reporter vector (pEGFP-CA13). Gastric cancer AGS cells transfected with pEGFP-CA13 were cocultured with H. pylori or Escherichia coli. The numbers of green fluorescent protein (GFP)-positive cells were determined, and GFP, hMSH2, and hMLH1 protein levels were measured by Western blot. The effect of H. pylori on CpG methylation status of hMLH1 was determined by methylation-specific polymerase chain reaction. RESULTS: GFP levels and GFP-positive cell numbers in AGS cells cocultured with H. pylori significantly increased, as the levels of hMLH1 and hMSH2 dropped. H. pylori cocultures induced low-level CpG methylation of the hMLH1 promoter. Sequence analysis of cells cocultured with H. pylori showed an increased number of frameshift mutations and point mutations as compared to cells not cocultured with H. pylori (p = .03 and p = .001, respectively). CONCLUSIONS: This is the first report showing that H. pylori bacteria may lead to accumulation of genomic mutations, independently of underlying inflammation. This is associated with reduced DNA mismatch repair, and is at least in part associated with CpG methylation of the hMLH1 promoter. These data support the notion that H. pylori-induced mutations and epigenetic alterations in gastric epithelial cells during chronic gastritis may contribute to an increased risk of gastric cancer associated with H. pylori infection.     

白扁豆多糖对人胃癌细胞凋亡的作用及其机制

目的：探讨白扁豆多糖对人胃癌细胞凋亡的作用及其相关机制。方法：胃癌细胞HGC-27和SGC-7901经终浓度为16、8、4、2、1和0 μg/ml的白扁豆多糖作用24、48和72h,各设3个复孔。MTS法检测其增殖活性;分别取经4、0 μg/ml白扁豆多糖作用24h的HGC-27和SGC-7901细胞（各3个复孔）,JC-1染色观察线粒体膜电位,流式细胞仪分析细胞周期和凋亡率,QPCR法探讨Bcl-2、caspase-3和Bax基因的mRNA转录水平。结果：白扁豆多糖作用后,HGC-27和SGC-7901细胞线粒体膜电位降低;细胞增殖显著受抑制（P<0.01）,且效果与药物作用浓度和时间有关;细胞凋亡率分别为53.15%和38.77%,均较PBS处理组（8.07%和6.03%）明显增加（P<0.01）,而细胞周期无显著变化;同时,细胞内Bcl-2基因的转录水平明显受抑制,Bax和caspase-3基因的转录明显上调（P<0.01）。结论：白扁豆多糖可通过调节Bax-Bcl-2-caspase3通路,诱导胃癌细胞HGC-27和SGC-7901凋亡。     

Low expression of ERK signaling pathway affecting proliferation,cell cycle arrest and apoptosis of human gastric HGC-27 cells line

This study was carried out for the first time to examine the potential role and the underlying mechanisms of Lycopene in the gastric cancer HGC-27 cells. HGC-27 cells were seeded onto heat-sterilized coverslips in six-well plates and exposed to Lycopene (5, 10, 20, 30 and 40 μmol/L) for periods of 72 h at 37 °C. Results showed that Lycopene (5, 10, 20, 30 and 40 μmol/L) dose-dependently increased NBT positive rate and decreased lactate dehydrogenase activity in HGC-27 cells. In addition, Lycopene (5, 10, 20, 30 and 40 μmol/L) inhibited proliferation and induced G0–G1 phase cell cycle arrest in HGC-27 cells. Western blot and FQRT-PCR analysis showed that Lycopene decreased pERK and extracellular signal-regulated kinase (ERK) protein and mRNA expression in a dose-dependent manner. These findings demonstrate that Lycopene inhibited gastric cancer HGC-27 cells growth and stimulated its apoptosis via the suppressing ERK signaling pathway.     

Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1). This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.     

Helicobacter pylori inhibits gastric cell cycle progression

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.     

Comparative immunoproteomics of identification and characterization of virulence factors from Helicobacter pylori related to gastric cancer

Helicobacter pylori is an important risk factor of gastric cancer (GC). Although many H. pylori virulence factors have been reported, the pathogenic mechanism by which H. pylori infection causes GC remains unclear. The aims of this study were to identify GC-related antigens from H. pylori and characterize their roles in the development of GC. As GC and duodenal ulcer (DU) are considered clinically divergent, we compared two-dimensional immunoblots of an acid-glycine extract of H. pylori probed with serum samples from 15 patients with GC and 15 with DU to find GC-related antigens, which were subsequently identified by mass spectrometry. Many protein spots were recognized by more than one serum, and 24 of these were better recognized by GC sera. The proteins showing higher frequency of recognition in GC group are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase alpha subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR, and membrane fusion protein. Of these proteins, GroES was identified as a dominant GC-related antigen with a much higher seropositivity of GC samples (64.2%, n = 95) compared with 30.9% for gastritis (n = 94) and 35.5% for DU (n = 124). GroES seropositivity was more commonly associated with antral GC than with non-antral GC (odds ratio = 2.7; 95% confidence interval, 1.1-6.7). In peripheral blood mononuclear cells, GroES stimulated production of interleukin (IL)-8, IL-6, granulocyte macrophage colony-stimulating factor, IL-1beta, tumor necrosis factor-alpha, cyclooxygenase-2, and prostaglandin E(2). Moreover when incubated with gastric epithelial cells, GroES induced expression of IL-8, cell proliferation, and up-regulation of c-jun, c-fos, and cyclin D1 but caused down-regulation of p27(Kip1). We conclude that GroES of H. pylori is a novel GC-associated virulence factor and may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation.     

Helicobacter pylori infection triggers aberrant expression of activation-induced cytidine deaminase in gastric epithelium

Infection with Helicobacter pylori (H. pylori) is a risk factor for the development of gastric cancer. Here we show that infection of gastric epithelial cells with 'cag' pathogenicity island (cagPAI)-positive H. pylori induced aberrant expression of activation-induced cytidine deaminase (AID), a member of the cytidine-deaminase family that acts as a DNA- and RNA-editing enzyme, via the IkappaB kinase-dependent nuclear factor-kappaB activation pathway. H. pylori-mediated upregulation of AID resulted in the accumulation of nucleotide alterations in the TP53 tumor suppressor gene in gastric cells in vitro. Our findings provide evidence that aberrant AID expression caused by H. pylori infection might be a mechanism of mutation accumulation in the gastric mucosa during H. pylori-associated gastric carcinogenesis.