Implication of Leptin-Signaling Proteins and Epstein-Barr Virus in Gastric Carcinomas

<div><p>We investigated the clinicopathological implications of leptin-signaling proteins and Epstein-Barr virus (EBV)-infection status in gastric carcinomas. Immunohistochemistry for leptin signalling-related proteins (leptin, leptin-receptor, pSTAT3, ERK, pAkt, mTOR and HIF-1 alpha), and <i>in situ</i> hybridization for EBV-encoded small RNAs was performed in 343 cases of gastric carcinomas. The siRNA against leptin-receptor was transfected into three stomach cancer cell lines, and western blot for caspase 3 was performed. The TNM stage was a prognostic factor in all 343 patients, and was negatively correlated with expression of leptin, pSTAT3, ERK, pAkt, mTOR and HIF-1 alpha (<i>P</i> < 0.05). Leptin-receptor expression was correlated with poor survival in 207 patients of the advanced gastric cancer (AGC) subgroup, 139 of the Lauren diffuse group, and in 160 patients with lymph node metastasis (<i>P</i> < 0.05, respectively). Additionally, in stomach cancer cells, cleaved caspase 3 level increased by leptin-receptor inhibition, that is, apoptosis increased. Interestingly, EBV-positive AGC (n = 29) tended to show better survival of patients than EBV-negative AGC (n = 178) (<i>P</i> = 0.06). pAkt expression was related with a good survival of 32 patients (9%) in the EBV-positive subgroup, but was not an independent prognostic factor. Among, leptin signaling-related proteins, expressions of leptin-receptor and mTOR were different between EBV-positive subgroup and EBV-negative subgroup (<i>P</i> < 0.05, respectively). In conclusion, leptin-signaling proteins and EBV status show different significance on patient survival, according to subsets of gastric carcinomas. The leptin-receptor may predict poor patient prognosis in the AGC, Lauren diffuse and lymph node metastasis subgroups, while EBV-positive status can show a good prognosis in the AGC. Each leptin signaling-related protein may be differently involved in carcinogenesis of EBV-negative and EBV-positive subsets.</p></div

Antibodies used for immunohistochemical staining.

<p>Antibodies used for immunohistochemical staining.</p

Overview of clinicopathological features and immunohistochemical staining results.

<p>^a<i>P</i> < 0.05. EBV, Epstein-Barr virus.</p><p>Overview of clinicopathological features and immunohistochemical staining results.</p

Kaplan-Meier survival curves in each group of gastric carcinomas according to leptin-receptor expression status (A-D).

<p>(<b>A</b>) Advanced TNM stage was associated with poor survival of patients in all cases of gastric carcinoma (n = 343) (<i>P</i> < 0.001). (<b>B</b>) Leptin-receptor positivity was related with unfavorable survival outcome in the advanced gastric carcinoma subgroup (n = 207) (<i>P</i> = 0.015). (<b>C</b>) Leptin-receptor positivity was correlated with poor survival rate in the diffuse-type gastric carcinoma subgroup (n = 139) (<i>P</i> = 0.007). (<b>D</b>) Leptin-receptor positivity was compatible with lower survival rate in patients with lymph node metastasis (n = 160) (<i>P</i> = 0.023).</p

Comparison of immunohistochemical features between EBV-positive gastric carcinomas and EBV-negative gastric carcinomas.

<p>EBV, Epstein-Barr virus; NS, not significant.</p><p>Comparison of immunohistochemical features between EBV-positive gastric carcinomas and EBV-negative gastric carcinomas.</p

Effect of leptin-receptor inhibition in gastric carcinoma cells.

<p>Leptin-receptor in total cell lysate was inhibited by siRNA against leptin-receptor (50 μM). Cleaved caspase 3 (p11, p17 and p20 subunits) was remarkably increased in gastric carcinoma cell lines transfected with siRNA against leptin-receptor, comparing to each original cell line and each scrambled siRNA-treated cell line. The beta-actin was loading controls. ‘Si scramble’ means cells transfected with scrambled siRNA, and ‘Si Leptin-R,’ cells transfected with siRNA against leptin-receptor.</p

Representative immunohistochemical features of seven protein markers including proteins related to leptin signaling pathway (A-G) and <i>in situ</i> hybridization of Epstein-Barr virus encoded small RNAs (H).

<p>(<b>A</b>) Leptin shows cytoplasmic expression in cancer cells (x 400). (<b>B</b>) Leptin-receptor exhibits membranous staining in cancer cells (x 200). (<b>C</b>) pSTAT3 shows nuclear staining in cancer cells (x 400). (<b>D</b>) ERK has nuclear positivity in cancer cells (x 400). (<b>E</b>) pAkt shows cytoplasmic or nuclear staining in cancer cells (x 200). (<b>F</b>) mTOR has cytoplasmic positivity in cancer cells (x 200). (<b>G</b>) HIF-1 alpha has cytoplasmic or nuclear staining in cancer cells (x 400). (<b>H</b>) Most of the cancer cells show blue colored nuclear staining on <i>in situ</i> hybridization for Epstein-Barr virus (x 400).</p

Histomorphological and Immunophenotypic Features of Pill-Induced Esophagitis

<div><p>The aim of this study was to investigate histomorphological and immunophenotypic features in pill-induced esophagitis. We comparatively evaluated the histomorphological, immunophenotypic features of pill-induced esophagitis vs. reflux esophagitis, as well as clinical information and endoscopic findings. Fifty-two tissue pieces from 22 cases of pill-induced esophagitis, 46 pieces from 20 reflux esophagitis, and 16 pieces from 14 control samples were subjected to immunohistochemistry for inflammatory infiltrates (CD3 for T lymphocyte, CD20 for B lymphocyte, CD56 for NK cell, CD68 for macrophage, CD117 for mast cell) and eosinophil chemotaxis-associated proteins (Erk, leptin, leptin receptor, pSTAT3, phospho-mTOR). As a result, Histomorphology showed that a diffuse pattern of dilated intercellular spaces was more frequently observed in pill-induced esophagitis, while reactive atypia and subepithelial papillary elongation were more often found in reflux esophagitis (<i>P</i> < 0.05, respectively). Interestingly, intraepithelial eosinophilic microabscess, intraepithelial pustule and diffuse pattern of dilated intercellular spaces were observed in 14% (3 cases), 9% (2 cases) and 32% (7 cases) of pill-induced esophagitis, respectively, but in no cases of reflux esophagitis. Regarding intraepithelial inflammatory infiltrates in pill-induced esophagitis, T lymphocytes were the most common cells, followed by eosinophil; 11 and 7 in one x400 power field, respectively. Intraepithelial pSTAT3-positive pattern was more frequently observed in pill-induced esophagitis than in reflux esophagitis, at 45% (10 cases) versus 10% (2 cases), respectively (<i>P</i> < 0.05). Considering the distal esophageal lesion only, intraepithelial pustule, diffuse dilated intercellular spaces and stromal macrophages were more frequently found in distal pill-induced esophagitis, whereas reactive atypia and intraepithelial mast cells in reflux esophagitis (<i>P</i> < 0.05, respectively). In conclusion, diffuse dilated intercellular spaces, intraepithelial eosinophil microabscess, pustule, T lymphocytes, eosinophils, and pSTAT3 positivity can be added to histopathological features of pill-induced esophagitis, other than non-specific ulcer. Besides, distal pill-induced esophagitis may be histopathologically differentiated from reflux esophagitis.</p></div

Antibodies used in immunohistochemistry.

<p>Antibodies used in immunohistochemistry.</p

Immunohistochemical microphotographs of esophageal mucosal tissue in pill-induced esophagitis.

<p>(A) CD3. T lymphocytes are lodged between squamous epithelial cells. (B) CD117. Intraepithelial mast cells are rarely found. (C) & (D) pSTAT3. Diffuse nuclear staining is exhibited in this case (C), but was negative in another case (D). (E) leptin receptor. This picture shows a negative pattern with normally stained basal cells. (F) phospho-mTOR. Membranous staining is noted in only two cells (at the right middle portion, and the left lower end). Magnifications: A, B, E, F, x400 and C, D x200 (immunohistochemical stain).</p