OBJECTIVETo investigate the synergistic effect between the N-terminus domain of the a2 isoform of vacuolar ATPase (a2NTD) and macrophage colony-stimulating factor (M-CSF) on modulating macrophage polarization and the impact of polarized macrophages on proliferation of gastric cancer cells.

METHODSPeripheral blood mononuclear cells were derived from healthy donor and induced into macrophages. Then macrophages were randomly divided into four groups: the control group (RPMI 1640), the experimental group I (M-CSF 100 μg/L), the experimental group II (a2NTD 500 μg/L) and the experimental group III (a2NTD 500 μg/L plus M-CSF 100 μg/L). After stimulation for 48 hours, double color immunofluorescence cytochemistry was adopted to detect the expression of cell membrane molecules on macrophages; ELISA was used to measure the secretion of cytokines IL-10 and IL-12; CCK-8 assay was used to evaluate the impact of macrophages on proliferation ability of gastric cancer cell strain SGC-7901.

RESULTSThe expression of CD68, also known as macrophage surface antigen, was detected on macrophage membrane in all four groups (+). The mean absorbance (A) was 0.092 ± 0.005 in control group, 0.095 ± 0.006 in group I, 0.094 ± 0.005 in group II, 0.094 ± 0.005 in group III, and no significant differences were observed among 4 groups (all P>0.05). Meanwhile, the expression of CD206, which mainly exists on M2 macrophage membrane, was hard to detect in control group (-) with A 0.025 ± 0.004; it was normal in groupI and group II (+) with A 0.191 ± 0.012 in group I and 0.197 ± 0.136 in group II (P=0.212), and it was up-regulated significantly in group III (+++) with A 0.285 ± 0.011. There were significant differences between either two groups except group I and group II (all P<0.01). Secretion of IL-10 in group I and group II [(85.65 ± 13.64) ng/L and (87.77 ± 14.25) ng/L] was significantly higher compared with control group [(71.67 ± 7.56) ng/L, P<0.01]. Secretion of IL-12 in group I and group II [(9.91 ± 1.50) ng/L and (10.15 ± 1.80) ng/L] was significantly lower compared with control group [(16.87 ± 1.10) ng/L, P<0.01]. Secretion of IL-10 in group III [(116.98 ± 14.27) ng/L] was the highest, and secretion of IL-12 [(5.31 ± 0.88) ng/L] was the lowest (all P<0.01). There was a synergistic effect between a2NTD and M-CSF on the secretion of both IL-10 and IL-12. Elevated proliferation of gastric cancer cell strain SGC-7901 was detected in all four groups, in which group III showed the greatest impact compared with other 3 groups (P<0.01).

CONCLUSIONSa2NTD and M-CSF show a synergistic effect in modulating macrophage phenotype and the secretion of IL-10 and IL-12. The polarized macrophage can significantly enhance proliferation of gastric cancer cell strain SGC-7901.

Cell Proliferation ; Humans ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Macrophage Colony-Stimulating Factor ; pharmacology ; Macrophages ; cytology ; Phenotype ; Stomach Neoplasms ; pathology ; Tumor Cells, Cultured ; Vacuolar Proton-Translocating ATPases ; pharmacology

NADPH oxidase produces a large amount of reactive oxygen species (ROS) in Helicobacter pylori (H. pylori)-induced gastric epithelial cells. Even though ROS mediate apoptotic cell death, direct involvement of NADPH oxidase on H. pylori-induced apoptosis remains unclear. Besides, H. pylori isolates show a high degree of genetic variability. The predominant genotype of H. pylori in Korea has been reported as cagA+, vacA s1b, m2, iceA genotype. Present study aims to investigate whether NADPH oxidase-generated ROS mediate apoptosis in human gastric epithelial AGS cells infected with H. pylori in a Korean isolate. AGS cells were pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) and cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Cell viability, hydrogen peroxide level, DNA fragmentation, and protein levels of p53, Bcl-2, and Bax were determined. Results showed that H. pylori inhibited cell viability with the density of H. pylori added to the cells. Inhibition of NADPH oxidase by DPI suppressed H. pylori-induced cell death, increased hydrogen peroxide, DNA fragmentation, and the ratio of Bax/Bcl-2, and p53 induction in AGS cells dose-dependently. The results suggest that targeting NADPH oxidase may prevent the development of gastric inflammation associated with H. pylori infection by suppressing abnormal apoptotic cell death of gastric epithelial cells.
Apoptosis ; Apoptosis Regulatory Proteins/metabolism ; Cell Survival ; Epithelial Cells/metabolism/microbiology ; Gastric Mucosa/metabolism ; Helicobacter Infections/*metabolism/microbiology ; Helicobacter pylori/drug effects/genetics/*isolation & purification ; Humans ; NADPH Oxidase/metabolism ; Onium Compounds/*antagonists & inhibitors/pharmacology ; Oxidative Stress/drug effects ; Reactive Oxygen Species/metabolism ; Republic of Korea ; Stomach/cytology/*metabolism/microbiology

Animals ; Apoptosis ; Cell Differentiation ; Cell Proliferation ; Fluoride Poisoning ; metabolism ; pathology ; Fluorosis, Dental ; metabolism ; pathology ; Hedgehog Proteins ; genetics ; metabolism ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteoclasts ; cytology ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Signal Transduction ; Stomach Neoplasms ; metabolism ; pathology

BACKGROUND/AIMS: Doublecortin and CaM kinase-like-1 (DCAMKL1) is a marker of stem cells expressed predominantly in the crypt base in the intestine. However, DCAMKL1-positive cells have been shown to be differentiated tuft cells rather than quiescent progenitors. Tuft cells are the only epithelial cells that express cyclooxygenase 2 (COX-2) in the normal intestinal epithelium. We previously generated Cdx2-transgenic mice as model mice for intestinal metaplasia and gastric carcinoma. In the current study, we investigated the association between COX-2 and DCAMKL1 in gastric carcinoma. METHODS: We examined the association between COX-2 and DCAMKL1 expression in gastric carcinomas in clinical samples (early gastric well-differentiated adenocarcinoma) and Cdx2-transgenic mice; and the DCAMKL1-transgenic mouse stomach using immunohistochemistry and quantitative real-time polymerase chain reaction. RESULTS: The COX-2-expressing cells were scattered, not diffusely expressed, in gastric carcinomas from humans and Cdx2-transgenic mice. DCAMKL1-positive cells were also scattered in the gastric carcinomas, indicating that tuft cells could still be present in gastric carcinoma. COX-2 was expressed in DCAMKL1-positive tuft cells in Cdx2- and DCAMKL1-transgenic mouse stomachs, whereas the Sox9 transcription factor was ubiquitously expressed in gastric carcinomas, including COX-2-positive cells. CONCLUSIONS: COX-2 is expressed in DCAMKL1-expressing quiescent tuft cells in gastric carcinoma.
Adenocarcinoma/metabolism ; Animals ; Cyclooxygenase 2/genetics/*metabolism ; Epithelial Cells/metabolism ; Gastric Mucosa/metabolism ; Humans ; Intestinal Mucosa/cytology/*enzymology/metabolism ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Mice ; Mice, Transgenic ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; SOX9 Transcription Factor/genetics/metabolism ; Stomach Neoplasms/*enzymology/genetics

OBJECTIVETo dynamically investigate the morphology of human gastric cancer SGC-7901 cell clones, and then compare the tumorigenic ability of different clones in order to identify the tumor stem cell clones.

METHODSClones derived from gastric cancer SGC-7901 cells were assessed by morphological observation, and the clone formation rate and proportion of each clone were calculated. The expression of CD44 and CDX2 in different clones was detected by immunofluorescence microscopy and Western blot. Furthermore, different clones were isolated and cultured, and their self-renewal property was assayed. Cells of different clones were subcutaneously inoculated into nude mice and the tumorigenic ability of each group was determined.

RESULTSClones derived from gastric cancer SGC-7901 cells had three types, i.e. clones of tight, transitional and loose types. The total clone formation rate was (9.80 ± 1.07)%, and the proportion of tight, transitional and loose type clones was 10.2%, 56.0% and 33.8%, respectively. The results of immunofluorescence microscopic examination showed that the signal of CD44 was significantly stronger in the tight clones than in the transitional and loose clones, however, the signal of CDX2 was weakest in the tight colonies. The results of Western blot were consistent with that of immunofluorescence microscopic observation. SGC-7901 cells of tight clones possessed strong ability of self-renewal and in vivo tumorigenicity in the nude mice.

CONCLUSIONSGC-7901 cell clones vary in morphology and differentiation, and the tight type clones may include rich gastric cancer stem cells.

Animals ; CDX2 Transcription Factor ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Clone Cells ; classification ; Female ; Homeodomain Proteins ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplastic Stem Cells ; cytology ; metabolism ; Random Allocation ; Stomach Neoplasms ; metabolism ; pathology

OBJECTIVETo study the inhibition and killing effect of transgenic LIGHT umbilical cord blood mesenchymal stem cells (UCBMSCs) on stomach carcinoma.

METHODSThe LIGHT gene was recombined to construct the transfer plasmid pGC-FU-LIGHT by infusion technique. The 293T cells were co-transfected with the transfer plasmid pGC-FU-LIGHT, the construction plasmid Helper 1.0 and the envelope plasmid Helper 2.0 with the help of lipofectamine 2000 to produce lentiviral particles. Transgenic UCBMSCs(MSC-LIGHT) and empty carrier UCBMSCs (MSC) were obtained. Human gastric cancer cell SGC-7901 was injected into nude mice subcutaneously groin. The model of transplanted human gastric cancer cell SGC-7901 in nude mice was established. Tumorigenesis nude mice were separated into three groups randomly with 5 in each group: MSC-LIGHT group, MSC group, and NS group. Three groups of nude mice were injected around the tumor with MSC-LIGHT, MSC and NS every other day for 3 times. Four weeks later, the transplanted gastric cancer volume was measured. The expressions of LIGHT in the three groups were determined by RT-PCR and ELISA method. The necrosis area in the tumors was calculated under pathological examination.

RESULTSThe average volume of transplanted tumor was(0.45±0.25) cm(3) in MSG-LIGHT group, (0.64±0.36) cm(3) in MSG group, and(1.21±0.79) cm(3) in NS group, and the difference was statistically significant(P<0.05). The LIGHT mRNA was 2.96±0.27, 1.23±0.47, and 0.73±0.10 respectively. The LIGHT protein was(167.89±2.31), (73.22±5.74), and (49.66±5.25) ng/L. The differences were all statistically significant among the three groups(both P<0.01). Pathological examination showed that the necrosis area was largest in MSC-LIGHT group.

CONCLUSIONTransgenic UCBMSCs secret LIGHT in a paracrine manner, which has inhibition and killing effects on stomach carcinoma.

Animals ; Cell Line, Tumor ; Fetal Blood ; cytology ; Genetic Therapy ; Humans ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plasmids ; genetics ; Stomach Neoplasms ; metabolism ; pathology ; therapy ; Transfection ; Tumor Necrosis Factor Ligand Superfamily Member 14 ; genetics ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate if granulocyte-macrophage colony stimulating factor (GM-CSF) gene-modified dendritic cells (DC) enhance antitumor immunity in vitro.

METHODSMice were injected with chemokine ligand 3 (CCL3) via the tail vein. Fresh B220(-)CD11c(+) cells were sorted from the peripheral blood mononuclear cells (PBMCs) and cultured into DCs by cytokines.DCs were transfected with AdGM-CSF gene at different ratios of multiplicity of infection (MOI) to determine the optimal gene transfection conditions, and the expression of GM-CSF was detected after transfection. The variation of GM-CSF gene-modifiedDCs were analyzed by morphological examination, phenotype analysis, and mixed lymphocyte reaction (MLR).DCs were loaded with gastric cancer antigen obtained by freezing and thawing method. The killing effect of DCs vaccine-stimulated T lymphocytes on gastric cancer cells was assessed by MTT assay. INF-gamma production was determined with the INF-gamma ELISA kit.

RESULTSB220(-)CD11c(+) cells increased obviously after CCL3 injection. The ELISA results showed that after GM-CSF gene modification, DCs could produce high level of GM-CSF. When DCs were transfected with AdGM-CSF gene at MOI equal to 100, the GM-CSF level in culture supernatants reached saturation [(130.00 +/- 12.61) pg/ml]. After GM-CSF gene-modification, DCs tend to be more maturated as detected by morphological observation and phenotype analysis. At the same time, the capacity of activating the proliferation of allogeneic T lymphocytes was enhanced greatly. T lymphocytes stimulated by DCs transfected with GM-CSF gene showed a specific killing effect on gastric carcinoma cells and produced high level of INF-gamma [(1245.00 +/- 13.75) pg/ml].

CONCLUSIONAfter GM-CSF gene modification, DCs can produce high level of GM-CSF, which tend to be more maturated, and the capacity of activating the proliferation of allogeneic T lymphocytes is enhanced greatly. GM-CSF gene modified DCs can induce specific CTL to target tumor cells in vitro.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Interferon-gamma ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; Stomach Neoplasms ; immunology ; metabolism ; pathology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

CpG-island margins and non-island-CpG sites round the transcription start sites of CpG-island-positive and -negative genes are methylated to various degrees in a tissue-specific manner. These methylation-variable CpG sites were analyzed to delineate a relationship between the methylation and transcription of the tissue-specific genes. The level of tissue-specific transcription was estimated by counting the number of the total transcripts in the SAGE (serial analysis of gene expression) database. The methylation status of 12 CpG-island margins and 21 non-island CpG sites near the key tissue-specific genes was examined in pluripotent stromal cells obtained from fat and bone marrow samples as well as in lineage-committed cells from marrow bulk, stomach, colon, breast, and thyroid samples. Of the 33 CpG sites examined, 10 non-island-CpG sites, but none of the CpG-island margins were undermethylated concurrent with tissue-specific expression of their nearby genes. The net methylation of the 33 CpG sites and the net amount of non-island-CpG gene transcripts were high in stomach tissues and low in stromal cells. The present findings suggest that the methylation of the non-island-CpG sites is inversely associated with the expression of the nearby genes, and the concert effect of transitional-CpG methylation is linearly associated with the stomach-specific genes lacking CpG-islands.
Adipose Tissue/cytology ; Adolescent ; Adult ; Adult Stem Cells/cytology/*metabolism ; Aged ; CpG Islands/*genetics ; *DNA Methylation ; Female ; Gene Expression Profiling ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Stomach/cytology/*metabolism ; Stromal Cells/metabolism ; Transcription Initiation Site ; Transcription, Genetic

OBJECTIVETo investigate the effect of Hedgehog (HH) pathway on proliferation and in vitro tumorigenicity of gastric cancer cell lines.

METHODSThe expression of SHH, PTCH, SMO, SUFU and GLI1 in seven cell lines were tested by RT-PCR. siRNA targeting GLI1 mRNA was transfected into MKN28 cells. Cell proliferation and in vitro tumorigenicity were examined by CCK8 and soft agar colony formation test.

RESULTSSHH in six gastric cancer cell lines was up-regulated. Expression of PTCH in KATOIII cell lines and expression of SUFU in MKN28 and KATOIII were reduced. GLI1 siRNA significantly inhibited the expression of GLI1 in MKN28 cell line. Growth rate and colony formation rate of MKN28 cells treated with GLI1 siRNA were significantly lower than those of control cells (all P <0.001).

CONCLUSIONSHH signaling pathway is widely activated in gastric cancer cell lines. The activation of HH signaling pathway promotes the growth of MKN28 cells.

Cell Line, Tumor ; Cell Proliferation ; Gastric Mucosa ; cytology ; Hedgehog Proteins ; metabolism ; Humans ; Oncogene Proteins ; metabolism ; RNA, Small Interfering ; Signal Transduction ; Stomach Neoplasms ; metabolism ; pathology ; Trans-Activators ; metabolism ; Zinc Finger Protein GLI1

OBJECTIVETo investigate the anti-gastric carcinoma immunological efficacy of dendritic cells (DC) precursors, that were mobilized into the peripheral blood by injection of macrophage inflammation protein-1 alpha (MIP-1 alpha), and induced by DC vaccine expressing melanoma antigen gene-3 (MAGE-3) ex vivo and in vivo.

METHODS615 mice were injected with MIP-1 alpha via the tail vein. Freshly isolated B220(-) CD11c+ cells were cultured with cytokines and assayed by phenotype analysis and mixed lymphocyte reaction (MLR). For adenoviral (Ad)-mediated gene transduction, cultured B220(-) CD11c+ cells were incubated with Ad-melanoma antigen gene-3. MIP-1 alpha-mobilized B220(-) CD11c+ cells pulsed MFC cells tumor lysate were used as positive control. The stimulated DC vaccination-induced T lymphocytes, and the killing effect of the T cells on gastric carcinoma cells were assayed by MTT. INF-gamma production was determined with the INF-gamma ELISA kit. To establish the solid tumor model, groups of 615 mice were injected with MFC cells subcutaneously into the abdominal wall. MIP-1 alpha-mobilized DC vaccines expressing MAGE-3 gene were used to immunize the mice after the challenge of MFC cells, then the tumor size and the survival of mice were examined to detect the therapeutic effect of DC vaccines.

RESULTSB220(-) CD11c+ cells increased obviously after MIP-1 alpha injection, and freshly isolated B220(-) CD11c+ cells cultured with mGM-CSF, IL-4, and mTNF-alpha were phenotypically identical to typical DC, gained the capacity to stimulate allogeneic T cells. These MIP-1 alpha-mobilized DCs were transduced with Ad-MAGE-3, which were prepared for DC vaccines expressing tumor antigen. T lymphocytes stimulated with DC-transduced with Ad-MAGE-3 showed specific killing effect on gastric carcinoma cells and produced high levels of INF-gamma [(1460.00 +/- 16.82) pg/ml]. Five days after the MFC cells challenge, the mice were subsequently injected with DC vaccines. The tumor size of the experimental group was significantly smaller than that in the positive control group and the negative control groups (P<0.01). Kaplan-Meier survival curves showed the survival of the experimental group mice was significantly longer than that of the control groups (P<0.01).

CONCLUSIONB220(-) CD11c+ DC precursors are rapidly accumulated in the peripheral blood after injection of MIP-1 alpha into mice, which can further differentiate into mature DCs. These MIP-1 alpha-mobilized DCs, when transduced with MAGE-3 gene, can induce specific CTL to gastric carcinoma cells ex vivo, and can generate anti-tumor therapeutic effects on MFC cells loading mice in vivo. The efficiency of anti-tumor therapeutic immunity induced by MIP-1 alpha-mobilized DCs expressing tumor antigen are much more potent than MIP-1 alpha mobilized DCs pulsed MFC cells tumor lysate.

Adenoviridae ; genetics ; Animals ; Antigens, Neoplasm ; genetics ; metabolism ; Cancer Vaccines ; therapeutic use ; Cell Line, Tumor ; Cell Proliferation ; Chemokine CCL3 ; metabolism ; pharmacology ; Dendritic Cells ; metabolism ; Female ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Neoplasm Proteins ; genetics ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; therapy ; T-Lymphocytes ; cytology ; metabolism ; T-Lymphocytes, Cytotoxic ; cytology ; metabolism ; Transduction, Genetic