Gastric Cancer Cell Tumor Research Articles

Surface Antigen Profiling of Helicobacter pylori-Infected and -Uninfected Gastric Cancer Cells Using Antibody Microarray.

Comprehensive immunophenotyping cluster of differentiation (CD) antigens in gastric adenocarcinoma, specifically between Helicobacter pylori-infected and -uninfected gastric cancer patients by using DotScan(™) antibody microarray has not been conducted. Current immunophenotyping techniques include flow cytometry and immunohistochemistry are limited to the use of few antibodies for parallel examination. We used DotScan(™) antibody microarray consisting 144 CD antibodies to determine the distribution of CD antigens in gastric adenocarcinoma cells and to elucidate the effect of H. pylori infection toward CD antigen expression in gastric cancer. Mixed leukocytes population derived from gastric adenocarcinoma patients were immunophenotyped using DotScan(™) antibody microarray. AGS cells were infected with H. pylori strains and cells were captured on DotScan(™) slides. Cluster of differentiation antigens involved in perpetuating the tolerance of immune cells to tumor cells was upregulated in gastric adenocarcinoma cells compared to normal cells. CD279 which is essential in T cells apoptosis was found to be upregulated in normal cells. Remarkably, H. pylori-infected gastric cancer patients exhibited upregulated expression of CD27 that important in maintenance of T cells. Infection of cagA+ H. pylori with AGS cells increased CD antigens expression which involved in cancer stem cell while cagA- H. pylori polarized AGS cells to express immune-regulatory CD antigens. Increased CD antigens expression in AGS cells infected with cagA+ H. pylori were also detected in H. pylori-infected gastric cancer patients. This study suggests the tolerance of immune system toward tumor cells in gastric cancer and distinct mechanisms of immune responses exploited by different H. pylori strains.

Long intergenic non-coding RNA 00152 promotes tumor cell cycle progression by binding to EZH2 and repressing p15 and p21 in gastric cancer.

Long noncoding RNAs (lncRNAs) play important regulatory roles in several human cancers. Integrated analysis revealed that expression of long intergenic non-coding RNA 152 (LINC00152) was significantly upregulated in gastric cancer (GC). Further analysis in a cohort of 97 GC patients revealed that LINC00152 expression was positively correlated with tumor invasion depth, lymph node metastasis, higher TNM stage, and poor survival. Gene set enrichment analysis revealed that cell proliferation and cell cycle progression were increased in patients with high LINC00152 expression. In both GC cell lines and xenograft systems, LINC00152 overexpression facilitated GC cell proliferation by accelerating the cell cycle, whereas LINC00152 knockdown had the opposite effect. Moreover, by binding to enhancer of zeste homolog 2 (EZH2), LINC00152 promotes GC tumor cell cycle progression by silencing the expression of p15 and p21. These findings suggest that LINC00152 may play contribute to the progression of GC and may be an effective therapeutic target.

Detection and cultivation of circulating tumor cells in gastric cancer.

Circulating tumor cells (CTCs) are important targets for treatment and critical surrogate markers when evaluating cancer prognosis and therapeutic response. A sensitive methodology for detecting CTCs in gastric cancer (GC) patients is needed. In this study we demonstrate a device for enrichment and cultivation of CTCs. In total, 22 patients with GC, all candidates for surgery, were enrolled in the study. Peripheral blood samples were collected before surgery, and patients were re-evaluated within operation and divided into two groups: resectable and non-resectable GC. A new size-based separation test for enrichment and cultivation of CTCs was used (MetaCell®). In addition to cytomorphological analysis, gene expression of tumor associated genes (Cytokeratin-18, Cytokeratin-19, Cytokeratin-20, Cytokeratin-7, EPCAM, MUC1, HER2, EGFR) and of leukocyte markers (e.g. CD45, CD68) was tested in enriched CTC fractions. CTCs were detected in 59 % of the patients studied (n = 13/22). CTCs were detected in seven patients of the resection group (7/10, 70 %) and six of the non-resectable group (6/12, 50 %). Enrichment of the viable CTCs allowed subsequent successful cultivation in vitro. The cytomorphological characterization of the CTCs was a prerequisite of random gene expression testing in CTC-positive samples. In CTC-positive samples gene expression of cytokeratin 18 and 19 was elevated in comparison to the whole blood gene expression analysis. CTCs were found to be present in both resectable and non-resectable gastric cancer patients. The size-based separation platform for CTCs may be used for in vitro cultivation, as well as in subsequent molecular analysis if desired. The sensitivity of CTC-detection could be enhanced by the combination of cytomorphological and molecular analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10616-015-9866-9) contains supplementary material, which is available to authorized users.

Overexpression of DEK in human gastric carcinoma and its clinicopathological significance.

75 Background: Gastric cancer (GAC) is the second most common cancer worldwide and the fifth leading cause of cancer-related death in Taiwan. To improve the survival of gastric cancer patients, biomarkers for early detection and effective anticancer therapy are required. Methods: Dysregulated proteins in GAC tumor cells were captured and identified by laser capture microdissection (LCM) and an isobaric tags for relative and absolute quantitation (iTRAQ)-labeling following basic-RP HPLC coupled to LTQ-orbitrap MS analysis. DEK, one of the most highly-expressed proteins in both intestinal and diffuse gastric cancers in iTRAQ analysis, was selected for further study. DEK proto-oncogene is originally identified as a fusion with the CAN nucleoporin protein in a subset of acute myeloid leukemia (AML) patients carrying the t(6;9) translocation, and DEK has been implicated in acute myeloid leukemia, melanoma, glioblastoma, hepatocellular carcinoma, and bladder cancer. The overexpression of DEK was confirmed using real-time quantitative-reverse transcription-polymerase chain reaction (Q-RT–PCR), Western blot, and immunohistochemical (IHC) analysis. Results: The data revealed that the expression of DEK mRNA was up-regulated in tumor tissues (compared with adjacent non-tumor tissues) of about 55.1% gastric patients. The expression of DEK protein was also up-regulated in tumor tissues identified by Western blot and IHC. To further understand the role of DEKgene in the human gastric cancer cells, the DEK knockdown stable clones were prepared in AZ521. The cells migration ability was reduced in DEK depletion stable clones, and the antitumor drug sensitivity and anoikis were increased in DEK depletion cells. Conclusions: These results indicate that DEK overexpression may contribute the poor prognosis of patients.

Benchmarking of gastric cancer sensitivity to anti-cancer drugs ex vivo as a basis for drug selection in systemic and intraperitoneal therapy.

BackgroundThe choice of drugs for treatment of advanced gastric cancer (GC) is empirical. The purpose of the current study was to benchmark ex vivo the sensitivity of GC tumor cells from patients to standard cytotoxic and some newly introduced targeted drugs (TDs), as a basis for drug selection in the treatment of GC.MethodsTumor cell samples from patients with GC were analyzed for sensitivity to 5-fluorouracil, cisplatin, oxaliplatin, irinotecan, mitomycin C, doxorubicin and docetaxel as well as for the targeted drugs bortezomib, sorafenib, sunitinib and rapamycin using a short-term in vitro assay based on retention of viable tumor cells of fluorescent fluorescein. Samples of normal mononuclear cells, chronic lymphocytic leukemia, ovarian cancer and colorectal cancer were included for comparison.ResultsThe GC samples were essentially as sensitive to the standard drugs and the TDs as those from colorectal cancer whereas the ovarian cancer samples were more sensitive. The individual GC samples varied considerably in sensitivity to increasing concentrations of the clinically used standard drugs. In GC, cisplatin was cross-resistant to oxaliplatin and 5-fluorouracil which, on the other hand, was not cross-resistant to the other cytotoxic drugs. The activity of sunitinib did not obviously correlate to that of the standard drugs.ConclusionEx vivo assessment of drug sensitivity of tumor cells from patients with GC is feasible and may provide information that could be useful for selection of drugs for treatment. Drug sensitivity varies considerably between and within individual samples arguing for individualized selection of drugs for chemotherapy.

Analysis of somatostatin receptors and somatostatin promoter methylation in human gastric cancer

Somatostatin (SST) is a gut peptide that is able to inhibit the growth of tumor cells in gastric cancer and other types of cancer. The present study investigated the mRNA and protein levels of SST and SST receptors (SSTRs) in human gastric cancer, and detected the DNA methylation of the SST promoter. The protein levels of SST were detected using a radioimmunoassay in 102 human gastric tissue specimens (51 pairs of samples from 51 gastric cancer patients, each pair of samples included a cancer tissue and a normal tissue sample). SST and SSTR mRNA expression was assessed by reverse transcription-PCR (RT-PCR), while SST promoter methylation was examined using quantitative methylation-specific PCR (qMSP) in 51 pairs of tissues. The association between SST protein and RNA levels and SST methylation and gastric cancer were also analyzed. The protein levels of SST were decreased in the gastric cancer group compared with those of the normal group (5.091±0.994 vs. 7.399±0.956 pg/mg; P<0.01). The RT-PCR analysis indicated that the mRNA levels of SST (0.218±0.183 vs. 0.456±0.331; P<0.001) and SSTRs in the gastric cancer group were lower compared with those of the normal gastric tissue group. The methylation proportion of SST was 45.1% (23/51) in the carcinoma group and 3.9% (2/51) in the normal group. In conclusion, SST promoter methylation is a common event in human gastric cancer and is connected with a decrease in SST protein and RNA levels and associated with gastric carcinogens.

P-0070 - Neoadjuvant Chemotherapy Within Combined Treatment Gastric Cancer Patients, Evaluation of Effectiveness

Background: The gastric cancer (GC), despite a steady decline in the incidence, is one of the most common malignancies in Ukraine and in other countries around the world. The major treatment strategy during the last decades has been postoperative systemic therapy. The results of published trials were reviewed. And neoadjuvant chemotherapy (CRT) was evaluated. Methods: Patients with GC (n = 55) randomly underwent neoadjuvant CRT (PELF) followed by planned surgical resection, group A (n = 34) or surgery alone, group B (n = 40) at clinic of National Cancer Institute of Ukraine, between 2009 and 2013. Overall survival (OS), local control (LC) and biological markers were estimated. Results: Correlations between expression levels of molecular marker VEGFR-1 and gastric cancer tumor cells sensitivity were found. It was no differences in 2-year overall survival rates (64% vs 56.6% for groups A and respectively, p = 0.22). Primary tumor volumetry showed difference between the average value before and after neoadjuvant CRT, 182 ± 11.4 mm3 vs 112 ± 8.7 mm3, p < 0.001. Conclusion: Performed analysis is an evidence of urgency and practical importance of the modern molecular technologies application for the purpose of stomach cancer patients treatment algorithms improvement. It was proved sensitivity for VEGFR-1 to chemotherapy in patients with GC. Determination of sensitivity to neoadjuvant CRT offers the prospect of achieving a reasonable treatment in patients with GC and reducing the overall cost of treatment.

A True Orthotopic Gastric Cancer Murine Model Using Electrocoagulation

Orthotopic mouse models of human gastric cancer represent an important invivo tool for testing chemotherapeutic agents and for studying intraluminal factors. Currently, orthotopic mouse models of gastric cancer require an operative procedure involving either injection or implantation of tumor cells in stomach layers. The resultant tumor does not grow from the stomach's mucosal surface, so it does not mimic the human disease process. A low-dose gastric mucosal coagulation was done transorally in the body of stomach using a specially designed polyethylene catheter in 16 female severe combined immunodeficient mice. This was followed by the instillation of SNU-16 human gastric cancer tumor cells (1×10(6) cells). Five mice each were euthanized at 1 and 2 months, and 6 mice were euthanized at 3 months. Three control mice underwent electrocoagulation alone and 3 mice underwent cell line instillation alone. Tumors were detected in 11 of 16 experimental mice, but not in the control mice. Tumors were noted in mice at 1 month. Over time, there was an increase in tumor growth and metastasis to lymph nodes and surrounding organs. Histopathologic evaluation showed that the tumors grew from the gastric mucosa. Our model is easy to create and overcomes the limitations of the existing models, as the tumor arises from the stomach's mucosal layer and mimics the human disease in terms of morphology and biologic behavior. This is the first report of a true orthotopic gastric cancer murine model. This model opens new doors for additional studies that were not possible earlier.

Inducible nitric oxide synthase expression correlates with angiogenesis, lymphangiogenesis, and poor prognosis in gastric cancer patients

Increased nitric oxide synthase expression plays a key role in tumor progression. To examine inducible nitric oxide synthase expression and its correlation with clinical variables, such as tumor progression, angiogenesis, lymphangiogenesis, and prognosis in gastric cancer, we studied inducible nitric oxide synthase expression in gastric cancer samples from 211 patients with 5-year follow-up. CD105 and D2-40 were adopted as biomarkers for tumor angiogenesis and lymphangiogenesis, respectively. Inducible nitric oxide synthase staining was mainly found in the cytoplasm of gastric cancer tumor cells. Positive inducible nitric oxide synthase immunoreactivity was seen in 54.03% of gastric cancer specimens, which was correlated with lymph node metastasis, vascular invasion, distant metastasis, and TNM stage. Compared with inducible nitric oxide synthase negative patients, inducible nitric oxide synthase-positive patients had significantly shorter survival times and higher microvessel density and lymphatic vessel density. Intratumor and peritumor blood microvessel density and lymphatic vessel density correlated with inducible nitric oxide synthase expression (Spearman ρ test, P < .05). We conclude that inducible nitric oxide synthase expression correlates with lymph node metastasis, vascular invasion, distant metastasis, TNM stage, and poor survival rate in gastric cancer. We propose that synthesized inducible nitric oxide synthase increases angiogenesis, and lymphangiogenesis thus promotes tumor progression. Inducible nitric oxide synthase expression may be a good biomarker for poor prognosis in gastric cancer.

Discovery and verification of matrix gla protein, a TNM staging and prognosis-related biomarker for gastric cancer

To analyze microarray datasets deposited in the public database and to identify TNM associated genes in gastric cancers. Microarray datasets of gastric cancer were selected from GEO database. Differentially expressed genes related to TNM staging were evaluated by significant analysis of the microarray using MultiExperiment Viewer (MEV) platform. Candidate gene expressions in gastric cancer tissues and cell lines were verified by reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR, Western blot and immunohistochemistry. GES4007 dataset was re-analyzed leading to the identification of 14 genes associated with TNM staging. Over-expression of matrix gla protein (MGP) was confirmed in gastric cancer cell lines and tumor tissues by quantitative RT-PCR, Western blot and immunohistochemistry. Increased MGP expression was found in 22 of 54 cases of (40.7%) gastric cancer specimens compared to the normal gastric tissues. The up-regulation of MGP mRNA expression closely correlated with TNM stage (P = 0.001) and prognosis (P = 0.006). Public databases of microarray studies are the valuable resources for data mining. MGP has been identified and confirmed as a novel biomarker for the TNM stage and prognosis of gastric cancer.

Limitations of Isolated Tumor Cells in Gastric Cancer: Heterogeneity Requests Systems Biology Approaches Towards Personalized Medicine

Limitations of Isolated Tumor Cells in Gastric Cancer: Heterogeneity Requests Systems Biology Approaches Towards Personalized Medicine

HLA‐class I expression in gastric cancer

We investigated the clinical impact of HLA-class I tumor cells in gastric cancer. HLA-class I expression was immunohistochemically evaluated in specimens from 141 gastric cancer patients. The correlation between HLA-class I expression and clinical factors was analyzed. HLA-class I was identified in 96 (68.1%) gastric carcinomas. The loss of HLA-class I significantly correlated with the depth of invasion (P < 0.01), nodal involvement (P < 0.05) and tumor histology (P < 0.01). According to the positivity of HLA-class I, shallow depth and the absence of nodal metastasis increased. HLA-class I expression was a significant prognostic factor in gastric cancer (P < 0.02); however, HLA-class I was not an independent prognostic factor by multivariate analysis. Our data may suggest that loss of HLA-class I in gastric cancer did not directly reflect immunological escape from tumor antigen-specific cytotoxic T lymphocytes, unlike in other cancers.

Detection of cancer cells disseminated in bone marrow using real-time quantitative RT-PCR of CEA, CK19, and CK20 mRNA in patients with gastric cancer

To determine the significance of bone marrow disseminated tumor cells in gastric cancer, we investigated the mRNA expression levels of carcinoembryonic antigen (CEA), cytokeratin 19 (CK19), and cytokeratin 20 (CK20) using the real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR). Bone marrow samples were aspirated from the sternum at the time of surgery in 65 patients with resectable gastric cancer. Total RNA was extracted from bone marrow; and the expression levels of CEA, CK19, and CK20 mRNA were determined by RQ-PCR using an ABI PRISM 7000 and quantified against the GAPDH mRNA level. The detection limits of these genes were determined in the gastric cancer cell line MKN-45 and the colon cancer cell line C-1, which had been serially diluted in peripheral blood mononuclear cells (PBMCs). A rate of 1 cancer cell/million PBMCs was obtained by detecting CEA and CK19 mRNA in MKN-45 and by detecting CK20 mRNA in C-1. In the clinical samples, only 1 of the 65 gastric cancer patients (1.5%) who had stage IV disease was positive for CEA, CK19, and CK20 mRNA; none of CEA, CK19, or CK20 mRNA was positive in the remaining 64 patients. No significant correlation was observed between disseminated cancer cells in bone marrow and clinicopathological features, including simultaneous or metachronous hepatic metastasis and patient survival. The incidence of disseminated cancer cells in bone marrow in our study appears low, unlike that in previous reports. The significance of disseminated cancer cells in bone marrow may also be quite low in gastric cancer.