Non-neoplastic Gastric Tissues Research Articles

LDH-Indomethacin Nanoparticles Antitumoral Action: A Possible Coadjuvant Drug for Cancer Therapy.

Indomethacin (INDO) has a mechanism of action based on inhibiting fatty acids cyclooxygenase activity within the inflammation process. The action mechanism could be correlated with possible anticancer activity, but its high toxicity in normal tissues has made therapy difficult. By the coprecipitation method, the drug carried in a layered double hydroxides (LDH) hybrid matrix would reduce its undesired effects by promoting chemotherapeutic redirection. Therefore, different samples containing INDO intercalated in LDH were synthesized at temperatures of 50, 70, and 90 °C and synthesis times of 8, 16, 24, and 48 h, seeking the best structural organization. X-ray diffraction (XRD), vibrational Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), spectrophotometric analysis in UV-VIS, and differential thermogravimetric analysis (TGA/DTA) were used for characterization. Our results indicate that higher temperatures and longer synthesis time through coprecipitation reduce the possibility of INDO intercalation. However, it was possible to establish a time of 16 h and a temperature of 50 °C as the best conditions for intercalation. In vitro results confirmed the cell viability potential and anticancer activity in the LDH-INDO sample (16 h and 50 °C) for gastric cancer (AGP01, ACP02, and ACP03), breast cancer (MDA-MB-231 and MCF-7), melanoma (SK-MEL-19), lung fibroblast (MRC-5), and non-neoplastic gastric tissue (MN01) by MTT assay. Cell proliferation was inhibited, demonstrating higher and lower toxicity against MDA-MB-231 and SK-MEL-19. Thus, a clinical redirection of INDO is suggested as an integral and adjunctive anticancer medication in chemotherapy treatment.

Immunoexpression of Trefoil Factor 1 in Non-Neoplastic and Neoplastic Canine Gastric Tissues.

Simple SummaryGastric carcinoma (GC) is the second leading cause of death in humans and the most frequent malignancy in the stomach of dogs. As in humans, the prognosis of canine gastric cancer is generally poor owing to the advanced stage at the time of diagnosis, resulting in limited treatment options. In dogs, the molecular mechanisms involved in the growth and progression of gastric cancer remain largely unknown. Trefoil factor 1 (TFF1) protein is a mucin-associated secretory molecule that plays an important role in the maintenance and protection of epithelial surface integrity. Some human studies showed that TFF1 can protect mucosa against damage and suppress carcinogenesis, while other studies showed that TFF1 can restrict cell adhesion, promote tumor cell invasion, and block necrosis of tumor cells. In human gastric cancer, TFF1 has been found to decrease, and it has been proposed that it might act as a tumor suppressor factor. The present study was carried out to investigate whether there is a relationship between TFF1 and canine gastric carcinogenesis. We found an association between reduced expression of TFF1 and the development and progression of gastric cancer in dogs. The pathological and behavioral similarities between spontaneous canine GC and human counterparts make it logical to assume that dogs may be a useful model for human gastric cancer.TFF1 expression is markedly reduced in human GCs, suggesting that TFF1 is a tumor suppressor for human gastric cancer. The present study evaluated the expression and distribution pattern of TFF1 in paraffin-embedded canine gastric tissue samples, including normal mucosa (n = 3), polyps (n = 8), carcinomas (n = 31) and their adjacent non-neoplastic mucosa (n = 30), neoplastic emboli (n = 14), and metastatic lesions (n = 9), by immunohistochemistry (IHC). All normal gastric tissues expressed TFF1 in the superficial foveolar epithelium and mucopeptic cells of the neck region. Most gastric polyps (GPs) displayed immunoreactivity for TFF1 in >75% of the epithelial component. In GCs, the expression of TFF1 was found reduced in 74.2% of the cases. The level of TFF1 expression had a decreased tendency from normal gastric mucosa to GPs and GCs (p < 0.05). No significant differences in the expression of TFF1 were found in GCs, according to age, sex, histological type based on World Health Organization (WHO) and Lauren classification, tumor location, depth of tumor invasion, presence of neoplastic emboli or metastatic lesions. The median survival time of GC patients with preserved and reduced TFF1 immunoexpression were 30 and 12 days, respectively. Kaplan–Meier analysis revealed no significant survival differences between the two groups (p > 0.05). These findings suggest that TFF1 protein may play a role in canine gastric carcinogenesis, and further studies are necessary to define its usefulness as a prognostic indicator in canine gastric carcinoma.

Virus–host interactions in carcinogenesis of Epstein-Barr virus-associated gastric carcinoma: Potential roles of lost ARID1A expression in its early stage

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a distinct molecular subtype of gastric cancer characterized by viral infection and cellular abnormalities, including loss of AT-rich interaction domain 1A (ARID1A) expression (lost ARID1A). To evaluate the significance of lost ARID1A in the development of EBVaGC, we performed in situ hybridization of EBV-encoded RNA (EBER) and immunohistochemistry of ARID1A in the non-neoplastic gastric mucosa and intramucosal cancer tissue of EBVaGC with in vitro infection analysis of ARID1A-knockdown and -knockout gastric cells. Screening of EBER by in situ hybridization revealed a frequency of approximately 0.2% EBER-positive epithelial cells in non-neoplastic gastric mucosa tissue samples. Six small foci of EBV-infected epithelial cells showed two types of histology: degenerated (n = 3) and metaplastic (n = 3) epithelial cells. ARID1A was lost in the former type. In intramucosal EBVaGC, there were ARID1A-lost (n = 5) and -preserved tumors (n = 7), suggesting that ARID1A-lost carcinomas are derived from ARID1A-lost precursor cells in the non-neoplastic mucosa. Lost ARID1A was also observed in non-neoplastic mucosa adjacent to an ARID1A-lost EBVaGC. In vitro experiments using siRNA knockdown and the CRISPR/Cas9-knockout system demonstrated that transient reduction or permanent loss of ARID1A expression markedly increased the efficiency of EBV infection to stomach epithelial cells. Taken together, lost ARID1A plays a role in initiating EBV-driven carcinogenesis in stomach epithelial cells, which develop to a distinct subtype of EBVaGC within the proper mucosal layer. Lost ARID1A is one of the constituents of virus-host interactions in the carcinogenesis of EBVaGC.

Detection of TERT Promoter Mutations Using Targeted Next-Generation Sequencing: Overcoming GC Bias through Trial and Error.

PurposeDetection of telomerase reverse transcriptase (TERT) promoter mutations is a crucial process in the integrated diagnosis of glioblastomas. However, the TERT promoter region is difficult to amplify because of its high guanine-cytosine (GC) content (> 80%). This study aimed to analyze the capturing of TERT mutations by targeted next-generation sequencing (NGS) using formalin-fixed paraffin-embedded tissues.Materials and MethodsWe compared the detection rate of TERT mutations between targeted NGS and Sanger sequencing in 25 cases of isocitrate dehydrgenase (IDH)-wildtype glioblastomas and 10 cases of non-neoplastic gastric tissues. Our customized panel consisted of 232 essential glioma-associated genes.ResultsSanger sequencing detected TERT mutations in 17 out of 25 glioblastomas, but all TERT mutations were missed by targeted NGS. After the manual visualization of the NGS data using an integrative genomics viewer, 16 cases showed a TERT mutation with a very low read depth (mean, 21.59; median, 25), which revealed false-negative results using auto-filtering. We optimized our customized panel by extending the length of oligonucleotide baits and increasing the number of baits spanning the coverage of the TERT promoter, which did not amplify well due to the high GC content.ConclusionOur study confirmed that it is crucial to consider the recognition of molecular bias and to carefully interpret NGS data.

Analysis of 8q24.21 miRNA cluster expression and copy number variation in gastric cancer.

Aim:To analyze gene expression and copy number of five miRNAs (miR-1204, miR-1205, miR-1206, miR-1207 and miR-1208) localized in this chromosome region in gastric cancer (GC). Materials& methods:65 paired neoplastic and non-neoplastic specimens collected from GC patients and 20 non-neoplastic gastric tissues from cancer-free individuals were included in this study. The expression levels of the five miRNAs were accessed by real time qPCR and were correlated. Results:MiR-1207-3p, miR-1205, miR-1207-5p and miR-1208 were upregulated in approximately 50% of GC tumors in relation to those of adjacent non-neoplastic tissues. MiR-1205 expression was associated with gain of gene copies and was upregulated in adjacent non-neoplastic samples relative to external controls. Conclusion: The coexpression of the 8q24 miRNAs indicated the role of miR-1205 in the initiation of gastric cancer development.

Feasibility Study on Discrimination of Neo-plastic and Non-Neoplastic Gastric Tissues Using Spark Discharge Assisted Laser Induced Breakdown Spectroscopy.

Introduction: The present work is a novel in vitro study that evaluated the possibility of diagnosing neoplastic from nonneoplastic gastric tissues using spark discharge assisted laser induced breakdown spectroscopy (SD-LIBS) method. Methods: In these experiments, the low energy laser pulses ablated a tiny amount of tissue surface leading to plasma formation. Then, a spark discharge was applied to plasma in order to intensify the plasma radiation. Light emission from plasma was recorded as spectra which were analyzed. Gastric tissues of 5 people were studied through this method. Results: The SD-LIBS technique had the potential to discriminate normal and cancerous tissues based on the significant differences in the intensities of some particular elements. The comparison of normalized calcium (Ca) and magnesium (Mg) peaks of neoplastic and nonneoplastic gastric tissues could be viewed as a practical measure for tissue discrimination since Ca and Mg peaks in spectra of neoplastic were noticeably higher than nonneoplastic. Conclusion: Considering the identification of gastric cancer, the applied method in these experiments seems quite fast, noninvasive and cost-effective with respect to other conventional methods. The significant increment of specific Ca and Mg lines of neoplastic gastric tissues in comparison to the nonneoplastic ones can be considered as valuable information that might bring about tissue classification. The number of samples in this work, however, was not sufficient for a decisive conclusion and further researches is needed to generalize this idea.

Neoangiogenesis of gastric submucosa-invasive adenocarcinoma.

Early gastric cancer may be defined as mucosal or submucosal invasive carcinoma, and exhibits a good prognosis: 90% of patients survive >10 years. Early gastric cancer infrequently exhibits lymph node metastasis, although submucosal invasion, the presence of vascular invasion and/or lymphatic permeation are independent risk factors for lymph node metastasis in early gastric cancer. The analysis of tumor lymphangiogenesis and angiogenesis are important to determine the extent of invasive progression and metastasis in patients. Previously, the presence of vessels expressing the D2-40 antibody and the factor-VIII protein has been identified immunohistochemically. The vessels that are immunoreactive for D2-40 and factor-VIII are morphologically similar to lymphatic vessels or small-size veins, also termed venules. In the present study, the association between tumor invasion and neoangiogenesis in early gastric cancer was examined. The D2-40/factor-VIII double-stained vessel (DSV) density was analyzed, in addition to lymphatic and blood vessel (vein and artery) density, using 46 submucosa-invasive and 50 mucosal carcinomas, and 20 non-neoplastic gastric tissues. The lymphatic density and DSV density of submucosa beneath the carcinoma and submucosa of the surrounding region in submucosa-invasive carcinoma were significantly increased (P<0.001) in comparison with those in mucosal carcinoma or non-neoplastic gastric tissue. No significant difference was observed in blood vessel density between non-neoplastic gastric, mucosal carcinoma and submucosa-invasive carcinoma tissues other than that of mucosa. The present study suggests the potential for the presence of D2-40/factor-VIII DSV and the importance of this vessel for neoangiogenesis in early gastric cancer.

Novel Insights into Gastric Cancer: Methylation of R-spondins and Regulation of LGR5 by SP1.

Recently, it was shown that leucine-rich repeat-containing receptor 5 (LGR5)-expressing stem cells are the cellular origin of intestinal-type gastric cancer. The aim of our study was to uncover regulatory mechanisms of LGR5 expression in gastric mucosa and their implications for cancer development. Reporter assays identified an LGR5 promoter fragment, which is highly relevant for active LGR5 expression. Chromatin immunoprecipitation verified that SP1 is bound within this region, and reporter activity increased in SP1 transfected cells. Subsequently, the expression of R-spondins (RSPO1 and RSPO2), ligands of LGR5, was explored in neoplastic and nonneoplastic gastric tissue and gastric cancer cell lines. Using IHC, distinct spatial expression patterns of LGR5, RSPO1, and RSPO2 were found in nonneoplastic stomach mucosa and gastric cancer. RSPO expression was lower in gastric cancer compared with nonneoplastic mucosa on both the transcriptional (P = 0.003 for RSPO1 and P = 0.000 for RSPO2; n = 50) and the translational level. Methylation-specific PCR showed higher methylation levels of RSPO1/2 and reexpression of RSPOs in the gastric cancer cell lines MKN45 and MKN74 were induced by demethylating 5-aza-C treatment. Finally, expression patterns of LGR5 and RSPO were similar in gastric cancer.Implications: This report identifies a regulatory mechanism of LGR5 expression in gastric carcinogenesis, with SP1 as an important component of the transcriptional complex and LGR5 activity, which is modulated by its ligands RSPO1 and RSPO2, whose expression is modulated by methylation.Visual Overview: http://mcr.aacrjournals.org/content/15/6/776/F1.large.jpg. Mol Cancer Res; 15(6); 776-85. ©2017 AACR.

Abstract 394A: Phosphatidylcholine-34:2 and -36:4 have tumor suppressive function for gastric cancer

Abstract Imaging mass spectrometry (IMS) is an emerging technique that enables us the quantitation and visualization of a number of biomolecules including phospholipids. Because antibodies against lipid molecules are hard to generate, IMS is a useful approach for lipid biology. Our colleagues and we used IMS apparatus named “iMScope™ (Shimadzu)” to detect the cancer specific phospholipids in liver, colorectal, oral, and breast cancer. Here, to identify possible anti-cancer drug candidate(s) for GC with no side effects, we performed an imaging mass spectrometry screening using a panel of non-neoplastic and neoplastic gastric tissue. Two species of phosphatidylcholine (PC), PC-36:4 and PC-34:2, were highly downregulated in GC. PC-36:4 and PC-34:2 suppressed the transformation of NIH3T3 cells driven by K-rasV12 and growth of 4 out of 8 GC cell lines, while the growth of non-transformed NIH3T3 was not affected by these lipids. Tumor growth of GC cells was also inhibited in nude mice on administration (i.v.) of these lipids without any side effects in vivo. Phosphorylation of Akt was downregulated by these lipids in some GC cell lines. In an immunohistochemical analysis, the expression of a PC-36:4 and PC-34:2 producing enzyme, LPCAT3, was diminished in GC tissue, and the low expression level of LPCAT3 was a predictor of poor survival of GC patients. Considering that PCs generally have no toxic effect for human, these data indicate the usefulness of PC-36:4 and PC-34:2 as potential anti-cancer drugs of GC. Stating the biochemical background of this observation, phospholipids are constituents of lipid bilayer and consist of a glycerol backbone, two esterified acyl chains, and a polar head group; phospholipids are classified into five subclass by the head group: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), or phosphatidylglycerol (PG). The Lands’ cycle produces the diversity of acyl chains’ compositions in phospholipids by phospholipase A2 and lysophospholipid acyltransferases. Recently, novel regulatory mechanisms of Akt activation via phospholipids were reported. For example, PS can bind Akt to induce conformational change, which facilitates the activation of Akt. On the other hand, a specific species of PC suppresses the interaction of Akt to phosphatidylinositol-3,4,5-trisphosphate to inhibit the downstream signaling. Because other specific phospholipids show the physiological functions in lipogenesis, fatty acid use, hepatic steatosis, and diabetes, the combination of the acyl chains are thought to define the functional properties of PCs. In these contexts, our data first disclosed that a specific PC plays a tumor suppressing role and possibly be a anti-cancer agent with minimal side-effect. Furthermore, our in vitro analysis was mechanistically rational in terms of the Akt-centered pathway of carcinogenesis. Citation Format: Nobuya Kurabe, Masako Suzuki, Yusuke Inoue, Tomoaki Kahyo, Moriya Iwaizumi, Hiroyuki Konno, Mitsutoshi Setou, Haruhiko Sugimura. Phosphatidylcholine-34:2 and -36:4 have tumor suppressive function for gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 394A.

Prognostic implications of phosphatidylinositol 3-kinase/AKT signaling pathway activation in gastric carcinomas.

IntroductionActivation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway plays a critical role in carcinogenesis and resistance to anticancer drugs. In this study, gastric carcinomas (GC) were investigated and statistical analyses were performed concerning the correlation between the clinicopathological features and activation of the PI3K/AKT pathway.Material and methodsImmunohistochemistry for p-AKT, p-mTOR and PTEN was performed in 239 GC and 200 non-neoplastic gastric tissues. The clinicopathological parameters were recorded from the medical charts. Statistical significance was defined by a p-value < 0.05.ResultsHigh p-AKT expression was observed in 10% of the normal gastric tissue and in 90% of GC, and it was significantly associated with tumor size (p < 0.001), T3/T4 tumors (p < 0.001), and presence of metastases (p = 0.02). Similarly, p-mTOR positivity was found in GC cells, but not in the normal gastric mucosa, and was correlated with perineural invasion (p = 0.02) and T3/T4 tumors (p = 0.03). On the other hand, PTEN expression was weak and focal in the tumor cells, while in the normal gastric tissue this staining was strong and diffuse. Importantly, the expression of p-mTOR and PTEN was associated with overall survival.ConclusionsThe results of the present study, characterized by the loss of PTEN expression and higher expression of p-AKT and p-mTOR in the majority of tumor cells, apparently are implicated in the carcinogenesis and progression of GC. The identification of p-mTOR and PTEN expression as prognostic factors corroborates the identification and use of potential target drugs that could be more efficient for the treatment of these patients.

Loss of ARID1A Expression is Related to Gastric Cancer Progression, Epstein-Barr Virus Infection, and Mismatch Repair Deficiency.

The AT-rich interactive domain 1A (ARID1A) gene encodes a member of the switch/sucrose nonfermentable (SWI-SNF) chromatin remodeling complex, and is considered to work as a tumor suppressor in concert with p53. We investigated the clinical significance of ARID1A protein expression in gastric cancer (GC), and examined its association with Epstein-Barr virus-associated (EBV) GC, mismatch repair (MMR) deficiency, and p53 alteration. We performed immunohistochemistry for ARID1A in 417 GC specimens using tissue microarray. EBV infection was examined using EBV-encoded small RNA in situ hybridization. Evaluation of MMR protein deficiency and p53 alteration was performed using immunohistochemistry, and microsatellite instability status was also assessed. Loss of ARID1A expression was observed in 21.1% of GC (88/417), but was not observed in gastric adenoma tissues or non-neoplastic gastric mucosa tissues. Loss of ARID1A showed positive correlations with advanced pTNM stage and tumor invasion (P=0.029 and 0.001, respectively). Overall survival was significantly influenced by the loss of ARID1A expression in wild-type p53 group (P=0.016, log-rank test). Moreover, ARID1A loss was significantly associated with EBV positivity, loss of MMR protein expression, and microsatellite instability high status (P=0.028, <0.001, and 0.011, respectively). All of the results from our cohort were verified using data from the Cancer Genome Atlas. In conclusion, loss of ARID1A is more common in advanced GC and is related to EBV positivity and MMR deficiency.

Overexpression of CX3CR1 is associated with cellular metastasis, proliferation and survival in gastric cancer.

The CX3CR1/CX3CL1 axis is involved in the metastasis and prognosis of many types of cancer; however, whether CX3CR1 is expressed in gastric cancer cells and whether it participates in gastric cancer metastasis remain unknown. We investigated the expression of CX3CR1 in gastric cancer tissues and non‑neoplastic gastric tissues in vivo and in gastric cancer cell lines and a gastric epithelial cell line in vitro, and then the functional roles of CX3CR1 in cellular metastasis, proliferation and survival were explored. We observed that CX3CR1 was highly expressed in gastric cancer tissues in vivo and was related to lymph node metastasis, higher clinical TNM stage and larger tumor size. In vitro, CX3CR1 overexpression promoted gastric cancer cell migration, invasion, proliferation and survival. Additionally, different from several chemokine receptors, CX3CR1 was also expressed in non-neoplastic gastric tissues and in gastric epithelial cells and played a functional role in vitro. Notably, gastric cancer tissues expressed higher CX3CR1 compared with that in the non-neoplastic gastric tissues in vivo, while in vitro, CX3CR1 expresssion in the gastric cancer cell lines was equivalent or significantly lower than that in the gastric epithelial cell line, which suggests that the high expression of CX3CR1 in gastric cancer in vivo might be induced, not constitutive. Altogether, our findings suggest that on the one hand overexpression of CX3CR1 promoted gastric cancer metastasis, proliferation and survival; on the other hand, appropriate expression of CX3CR1 in normal gastric tissues may play a physiological role in tissue remodeling after injury and/or epithelial renewal. Additionally, the tumor microenvironment may play an important role in the high expression of CX3CR1 in gastric cancer cells.

Analysis of a cytoskeleton-associated kinase PEAK1 and E-cadherin in gastric cancer

The expression of pseudopodium-enriched atypical kinase 1(PEAK1) has been studied in human cancers. However, their roles in gastric cancer are still unknown. In this study, gastric cancer tissue microarrays were constructed with 159 gastric cancer tissue samples, 150 non-neoplastic gastric epithelium specimens and 152 lymph node samples. Immunohistochemical staining for PEAK1 and E-cadherin was performed. Our study found negative expression of PEAK1 in 113 of 159 (71.1%) gastric cancers, in 46 of 150 (30.7%) non-neoplastic gastric epithelium tissues and in 69 of 94 (73.4%) metastatic lymph nodes. Negative expression of PEAK1 and E-cadherin associated with tumor grading, depth of invasion, lymph node metastases, pTNM stage and macroscopic type. Patients with either positive PEAK1 or E-cadherin expression had a significantly higher survival than those with negative expression. When combined, PEAK1−/E-cadherin− had a significantly poor prognosis than the rest of the patients. The expression of PEAK1 protein was positively correlated with E-cadherin in cancer tissues. Cox regression analyses showed that PEAK1, E-cadherin and PEAK1−/E-cadherin− were independent predictors of overall survival. In conclusion, our findings suggest that loss of PEAK1 may play an important role in carcinogenesis and development of gastric cancer through activating epithelial to mesenchymal transition.

Deregulated expression of Nucleophosmin 1 in gastric cancer and its clinicopathological implications

BackgroundThe process of gastric carcinogenesis still remains to be elucidated. The identification of genes related to this process may help to reduce mortality rates through early diagnosis and the development of new anticancer therapies. Nucleophosmin 1 (NPM1) acts in ribosome biogenesis, centrosome duplication, maintenance of genomic stability, and embryonic development. Recently, NPM1 has been implicated in the tumorigenesis processes. Here, we evaluated NPM1 gene and protein expression in gastric tumors and in corresponding non-neoplastic gastric samples.MethodsNPM1 protein expression was determined by Western blot in 17 pairs of gastric tumors and corresponding non-neoplastic gastric tissue. The protein immunoreactivity was observed in 12 tumor samples. mRNA expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in 22 pairs of gastric tumors and in matched non-neoplastic gastric tissue.ResultsNPM1 protein expression was significantly reduced in gastric cancer samples compared to matched non-neoplastic gastric samples (P = 0.019). The protein level of NPM1 was reduced at least 1.5-fold in 35% of tumors compared to paired non-neoplastic gastric tissue. However, NPM1 immunoreactivity was detected in neoplastic and non-neoplastic cells, including in intestinal metaplastic, gastritis and inflammatory cells. NPM1 was mainly expressed in nucleus and nucleolus subcellular compartments. The staining intensity and the percentage of immunoreactive cells varied among the studied cases. The NPM1 mRNA level was reduced at least 1.5-fold in 45.5% of samples and increased in 27.3% of samples. An inverse correlation between protein and mRNA expression was detected (r = -0.509, P = 0.037). Intestinal-type gastric cancer presented higher mRNA levels than diffuse-type (P = 0.026). However, reduced NPM1 protein expression was associated with intestinal-type gastric cancer compared to matched non-neoplastic gastric samples (P = 0.018). In addition, tumors from patients with known distant metastasis presented reduced NPM1 protein levels compared to tumors from patients without distant metastasis (P < 0.001).ConclusionAlthough the expression of NPM1 is heterogeneous in gastric tumors, our results suggest that NPM1 down-regulation may have a role in gastric carcinogenesis and may help in the selection of anticancer treatment strategies.

The distinct expression patterns of claudin-10, -14, -17 and E-cadherin between adjacent non-neoplastic tissues and gastric cancer tissues

BackgroundRecent data indicate that the cell adhesion proteins are abnormally regulated in several human cancers and the expression of the cell adhesion proteins E-cadherin and claudin proteins is involved in the etiology and progression of cancer. It is clear that these protein represent promising targets for cancer detection, diagnosis, and therapy.MethodsTo explore the expression distinction of the cell adhesion proteins claudin-10,-14,-17 and E-cadherin in the adjacent non-neoplastic tissues and gastric cancer tissues, 50 gastric cancer tissues and 50 samples of adjacent non-neoplastic tissues adjacent to the tumors were examined for expression of claudin-10,-14,-17 and E-cadherin by streptavidin-perosidase immunohistochemical staining method.ResultsThe positive expression rates of E-cadherin in gastric cancer tissues and adjacent non-neoplastic tissues were 32% and 74% respectively (P < 0.01). The positive expression rates of claudin-10 in gastric cancer tissues and adjacent non-neoplastic tissues were 24% and 72% respectively (P < 0.01). The positive expression rates of claudin-17 in gastric cancer tissues and adjacent non-neoplastic tissues were 18% and 70% (P < 0.01). In contrast, the positive expression rates of claudin-14 in gastric cancer tissues and adjacent non-neoplastic tissues were 58% and 24% respectively (P = 0.015 < 0.05) Thus in our study, the expression of E-cadherin, claudin-10, and claudin-17 was down-regulated in gastric cancer tissue while the expression of claudin-14 was up-regulated. Correlations between claudins and E-cadherin expression with lymphatic metastasis were observed.ConclusionOur study reveals that the expression of E-cadherin, claudin-10, and claudin-17 were down-regulated in gastric cancer tissue while the expression of claudin-14 was up-regulated and correlation between claudins and E-cadherin expression with lymphatic metastasis were observed.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1475928069111326.

Inducible nitric oxide synthase expression in gastric adenocarcinoma: impact on lymphangiogenesis and lymphatic metastasis

BackgroundLymphatic metastasis is the most important parameter in the spread of gastric carcinomas. Nitric oxide (NO) is a signaling molecule that plays an important role in inflammation and carcinogenesis. In this study, the possible link between inducible nitric oxide synthase (iNOS) expression with lymphangiogenesis and the clinicopathological parameters of gastric carcinomas was investigated.MethodsIn this study, iNOS expression and D2-40 (lymphatic endothelium-specific marker monoclonal antibody) reactivity were examined immunohistochemically in 41 gastric adenocarcinoma and 20 non-neoplastic gastric tissues. iNOS expression was scored semiquantitatively in the tumor parenchyma and stroma. D2-40-positive lymphatic vessels were used in the determination of lymphatic invasion and intratumoral and peritumoral lymphatic vascular density.ResultsiNOS expression was higher in gastric carcinoma tissue compared with non-neoplastic tissue. Particularly, iNOS expression in tumor cells was found to be closely related to lymphangiogenesis and lymphatic metastasis. The density of lymphatic invasion as well as intratumoral and peritumoral lymphatic vascular density were positively correlated with lymph node metastasis.ConclusionsOur results suggest that iNOS-mediated NO formation plays an important role in gastric carcinogenesis, tumor lymphangiogenesis, and the development of lymphatic metastases. Inhibition of the NO pathway may be an alternative treatment of gastric carcinomas.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1713572940104388.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

To evaluate the suitability of reference genes in gastric tissue samples and cell lines. The suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent non-neoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The ranking of the best single and combination of reference genes was determined by NormFinder, geNorm™, BestKeeper, and DataAssist™. In addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization. ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. The level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44). GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested.

Gastrokine 1 Expression in the Human Gastric Mucosa Is Closely Associated with the Degree of Gastritis and DNA Methylation

PurposeGastrokine 1 plays an important role in gastric mucosal defense. Additionally, the Gastrokine 1-miR-185-DNMT1 axis has been shown to suppress gastric carcinogenesis through regulation of epigenetic alteration. Here, we investigated the effects of Gastrokine 1 on DNA methylation and gastritis.Materials and MethodsExpression of Gastrokine 1, DNMT1, EZH2, and c-Myc proteins, and the presence of Helicobacter pylori CagA protein were determined in 55 non-neoplastic gastric mucosal tissue samples by western blot analysis. The CpG island methylation phenotype was also examined using six markers (p16, hMLH1, CDH1, MINT1, MINT2 and MINT31) by methylation-specific polymerase chain reaction. Histological gastritis was assessed according to the updated Sydney classification system.ResultsReduced Gastrokine 1 expression was found in 20 of the 55 (36.4%) gastric mucosal tissue samples and was closely associated with miR-185 expression. The Gastrokine 1 expression level was inversely correlated with that of DNMT1, EZH2, and c-Myc, and closely associated with the degree of gastritis. The H. pylori CagA protein was detected in 26 of the 55 (47.3%) gastric mucosal tissues and was positively associated with the expression of DNMT1, EZH2, and c-Myc. In addition, 30 (54.5%) and 23 (41.9%) of the gastric mucosal tissues could be classified as CpG island methylation phenotype-low and CpG island methylation phenotype-high, respectively. Reduced expression of Gastrokine 1 and miR-185, and increased expression of DNMT1, EZH2, and c-Myc were detected in the CpG island methylation phenotype-high gastric mucosa.ConclusionsGastrokine 1 has a crucial role in gastric inflammation and DNA methylation in gastric mucosa.

Absence of RKIP expression is an independent prognostic biomarker for gastric cancer patients

Gastric cancer is a leading cause of cancer-related mortality, and the presence of lymph node metastasis an important prognostic factor. Downregulation of RKIP has been associated with tumor progression and metastasis in several types of neoplasms, being currently categorized as a metastasis suppressor gene. Our aim was to determine the expression levels of RKIP in gastric tissues and to evaluate its impact in the clinical outcome of gastric carcinoma patients. RKIP expression levels were studied by immunohistochemistry in a series of gastric tissues. Overall, we analysed 222 non-neoplastic gastric tissues, 152 primary tumors and 42 lymph node metastasis samples. We observed that RKIP was highly expressed in ~83% of non-neoplastic tissues (including normal tissue and metaplasia), was lost in ~56% of primary tumors and in ~90% of lymph node metastasis samples. Loss of RKIP expression was significantly associated with several markers of poor clinical outcome, including the presence of lymph node metastasis. Furthermore, the absence of RKIP protein constitutes an independent prognostic marker for these patients. In conclusion, RKIP expression is significantly lost during gastric carcinoma progression being almost absent in lymph node metastasis samples. Of note, we showed that the absence of RKIP expression is associated with poor outcome features of gastric cancer patients, this being also an independent prognostic marker.

LARP7 is a potential tumor suppressor gene in gastric cancer

We previously reported frequent truncating mutations of the RNA-binding protein gene, La ribonucleoprotein domain family, member-7 (LARP7) in gastric cancers (GCs) with frequent microsatellite instability. LARP7 negatively regulates positive transcription elongation factor-b (p-TEFb) by binding to and stabilizing 7sk RNA. p-TEFb has been linked to proliferation and de-differentiation in various tissues. Therefore, we reasoned that loss of LARP7 may contribute to gastric tumorigenesis. In this study, we evaluated LARP7 mRNA expression in 18 GCs, their corresponding non-neoplastic gastric tissues (NGC), and 18 normal gastric tissues from healthy individuals (NN). We also assessed the effects of transient small interfering (siRNA)-mediated LARP7 knockdown in immortalized non-neoplastic gastric epithelial cells. LARP7 mRNA was significantly decreased in GCs (median 2.5) relative to NNs (median 14.9, P<0.01) as well as relative to their corresponding NGCs (median 8.1, P<0.01). Transfection of an siRNA directed against LARP7 (anti-LARP7 siRNA) into non-neoplastic gastric epithelial cells decreased 7sk levels by 72% relative to a control siRNA (P<0.01). Furthermore, anti-LARP7 siRNA transfection increased cell proliferation by 23% (P<0.01) and cell migration by 22% (P<0.001) relative to control siRNA transfection. Taken together, these findings suggest that LARP7 downregulation occurs early during gastric tumorigenesis and may promote gastric tumorigenesis via p-TEFb dysregulation.