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BTG (B-cell translocation gene) can inhibit cell proliferation, metastasis and angiogenesis, cell cycle progression, and induce differentiation in various cells. Here, we found that BTG3 overexpression inhibited proliferation, induced S/G2 arrest, differentiation, autophagy, apoptosis, suppressed migration and invasion in MKN28 and MGC803 cells (p < 0.05). BTG3 transfectants showed a higher mRNA expression of p27, Bax, 14-3-3, Caspase-3, Caspase-9, Beclin 1, NF-κB, IL-1, -2, -4, -10 and -17, but a lower mRNA expression of p21, MMP-9 and VEGF than the control and mock (p < 0.05). At protein level, BTG3 overexpression increased the expression of CDK4, AIF, LC-3B, Beclin 1 and p38 (p < 0.05), but decreased the expression of p21 and β-catenin in both transfectants (p < 0.05). After treated with cisplatin, MG132, paclitaxel and SAHA, both BTG3 transfectants showed lower viability and higher apoptosis than the control in both time- and dose-dependent manners (p < 0.05). BTG3 expression was restored after 5-aza-2'-deoxycytidine or MG132 treatment in gastric cancer cells. BTG3 expression was decreased in gastric cancer in comparison to the adjacent mucosa (p < 0.05), and positively correlated with venous invasion and dedifferentiation of cancer (p < 0.05). It was suggested that BTG3 expression might contribute to gastric carcinogenesis. BTG3 overexpression might reverse the aggressive phenotypes and be employed as a potential target for gene therapy of gastric cancer.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 3 is involved in calcium mobilization from the endoplasmic reticulum into the cytosol and is closely linked to metabolism, neuronal plasticity, gene transcription, cell growth, differentiation, apoptosis, protein folding and carcinogenesis. In order to elucidate the role of SERCA3 in colorectal carcinogenesis and subsequent progression, its expression was examined using immunohistochemistry and in situ hybridization (ISH) on tissue microarrays containing colorectal carcinomas, adjacent non-neoplastic mucosa (NNM) and adenoma, and metastatic carcinoma in lymph node and liver. Colorectal carcinoma tissue and cell lines were assessed for SERCA3 expression by western blotting or RT-PCR, respectively. SERCA3 was distinctively expressed in Colo201, Colo205, DLD-1, HCT-15, HCT-116, HT-29, KM-12, SW480, SW620 and WiDr cells at both the mRNA and protein levels. SERCA3 mRNA expression was low in carcinoma when compared to that in matched NNM by quantitative PCR (P<0.05), while the converse was true by ISH. Lower expression of SERCA3 was immunohistochemically observed in colorectal carcinoma when compared to that in NNM and adenoma (P<0.05). In contrast, primary carcinoma showed high SERCA3 expression when compared to that in metastatic carcinoma in lymph node or liver by IHC (P<0.05). Immunohistochemically, SERCA3 expression was negatively related to lymphatic invasion, but not with age, gender, depth of invasion, venous invasion, lymph node metastasis, distant metastasis, TNM stage, degree of differentiation or survival rate (P>0.05). There was a positive relationship between SERCA3 expression and serum CEA levels in the carcinoma patients (P<0.05). Cox's proportional hazards model indicated that depth of invasion and distant metastasis are independent prognostic factors for overall colorectal carcinomas (P<0.05). These findings suggest that aberrant SERCA3 expression is closely linked to the adenoma-adenocarcinoma sequence and progression of colorectal carcinomas.

B-cell translocation gene 3 (BTG3) is a member of the BTG family which inhibits cell proliferation, metastasis, and angiogenesis, and also regulates cell-cycle progression and differentiation in a variety of cell types. However, there is no study to analyze BTG3 expression in epithelial ovarian carcinoma (EOC). Here, we investigated the expression of BTG3 in EOC carcinogenesis and subsequent progression. BTG3 mRNA expression was detected by real-time RT-PCR in ovarian benign and malignant tumors. The expression of BTG3 protein was examined by immunohistochemistry on tissue microarrays containing ovarian normal tissue, benign and borderline epithelial ovarian tumors, and EOCs. Relationships of BTG3 with both EOC clinicopathology and prognosis were analyzed statistically. The expression of BTG3 protein was also evaluated in ovarian normal tissue, benign tumors, and EOCs by western blot. The BTG3 mRNA expression level was higher in ovarian normal tissue and benign tumors than that in borderline, primary, and metastatic carcinoma (p < 0.05), and was negatively correlated with dedifferentiation and FIGO staging of EOC (p < 0.05). Using western blot, BTG3 protein was found lower in EOCs compared to the normal and benign tumors (p < 0.05), and poorly differentiated EOCs showed lower BTG3 expression than well-differentiated and moderately differentiated EOCs (p < 0.05). Immunohistochemically, BTG3 protein expression was statistically lower in EOCs than normal tissue and benign tumors (p < 0.05). EOC patients with low BTG3 protein expression showed a higher incidence of metastasis (p = 0.020), poor differentiation (p = 0.030), and shorter disease-free time and overall survival time (p < 0.05). By using Cox's proportional hazard model, BTG3 protein expression and FIGO staging were independent prognostic factors for both disease-free time and overall survival time of EOCs (p < 0.05). It was suggested that down-regulated BTG3 expression might play roles in the pathogenesis and aggressiveness of EOC. BTG3 protein expression may be considered as a good marker to indicate the favorable prognosis of EOCs.

Beclin 1, an important autophagy-related protein in human cells, is involved in autophagy, differentiation, anti-apoptosis, and cancer progression, which is increased during periods of cell stress and extinguished during the cell cycle. In order to clarify the role of Beclin 1 in gastric carcinogenesis and subsequent progression, its expression was examined by immunohistochemistry and in situ hybridization (ISH) on tissue microarrays containing gastric carcinomas, adjacent non-neoplastic mucosa, and metastatic lymph node. Gastric carcinoma tissue and cell lines were studied for Beclin 1 expression by Western blot or RT-PCR, respectively. The results demonstrated that Beclin 1 was distinctively expressed in GES-1, AGS, BGC-823, GT-3 TKB, HGC-27, KATO-III, MGC-803, MKN28, MKN45, SCH, SGC-7901, or STKM-2 at both mRNA and protein levels. However, Beclin 1 mRNA was highly expressed in gastric carcinoma than matched mucosa by real-time PCR and ISH (P < 0.05). Beclin 1 expression was negatively related to distant metastasis and poor prognosis of gastric carcinoma (P < 0.05). Beclin 1 was highly expressed in male than female patients with gastric carcinoma (P < 0.05). The 65-year-elder patients with gastric carcinoma had higher Beclin 1 expression than the younger ones (P < 0.05). The diffuse-type carcinomas showed less Beclin 1 expression than intestinal- and mixed-type ones (P < 0.05). In intestinal-type gastric carcinoma, Beclin 1 expression was inversely associated with venous invasion, lymph node metastasis, and tumor-node-metastasis (TNM) staging (P < 0.05). Kaplan-Meier analysis indicated that Beclin 1 expression was positively linked to favorable prognosis of the patients with overall and intestinal-type carcinoma (P < 0.05). Cox's proportional hazard model indicated that venous invasion, lymph node metastasis, distant metastasis, TNM staging, and Beclin 1 expression were independent prognostic factors for gastric carcinomas (P < 0.05). It was suggested that aberrant Beclin 1 expression is closely linked to pathogenesis, metastasis, and differentiation of gastric carcinoma. Beclin 1 expression might be employed to indicate the favorable prognosis of gastric carcinomas as an independent factor.

Sarco (endo)plasmic reticulum Ca(2+)-ATPase (SERCAs) 3 is involved in calcium mobilization from endoplasmic reticulum into cytosol and closely links to metabolism, neuronal plasticity, gene transcription, cell growth, differentiation, apoptosis, protein folding, and carcinogenesis. To clarify the role of SERCA3 in gastric carcinogenesis and subsequent progression, its expression was examined by immunohistochemistry and in situ hybridization (ISH) on tissue microarrays containing gastric carcinomas, adjacent non-neoplastic mucosa (NNM), and metastatic lymph node. SERCA3 expression was studied in gastric carcinoma tissue and cell lines by Western blot, reverse transcriptase-polymerase chain reaction, or immunofluorescence. The results demonstrated that SERCA3 was distinctively expressed in GES-1, AGS, BGC-823, GT-3TKB, HGC-27, KATO-III, MGC-803, MKN28, MKN45, SCH, SGC-7901, and STKM-2 at both mRNA and protein levels. The carcinomas showed higher SERCA3 mRNA expression than the matched NNM by real-time PCR and ISH (P > 0.05). Immunohistochemically, SERCA3 expression was decreased from gastric NNM, primary to metastatic carcinoma (P > 0.05). SERCA3 expression was negatively related to depth of invasion, distant metastasis, and tumor node metastasis (TNM) staging (P > 0.05), but not to age, sex, lymphatic or venous invasion, or lymph node metastasis (P > 0.05). Kaplan-Meier analysis indicated that SERCA3 expression was positively associated with favorable prognosis of the patients with gastric carcinoma (P > 0.05). Cox's proportional hazard model indicated that venous invasion, distant metastasis and TNM staging (P > 0.05) were independent prognostic factors for gastric carcinomas. It was suggested that downregulated SERCA3 expression is closely linked to pathogenesis, invasion, metastasis, and prognosis of gastric carcinomas. It might be employed to indicate the pathobiological behaviors and prognosis of gastric carcinomas.

Glucose-related proteins (GRPs) are ubiquitously expressed in the endoplasmic reticulum and assist in protein folding and assembly, consequently considered to be molecular chaperones. GRP78 and GRP94 expression was induced by glucose starvation and up-regulated in samples taken from several different malignant tissues. To clarify the roles of both molecules in tumorigenesis and progression of colorectal carcinomas, immunohistochemistry (IHC) was performed on tissue microarrays containing colorectal carcinomas, adenomas and the non-neoplastic mucosa (NNM) using antibodies against GRP78 and GRP94. Their expression was correlated with the clinicopathological parameters of carcinomas. Both proteins were also studied in colorectal carcinoma cell lines (DLD-1, HCT-15, SW480 and WiDr) by IHC and Western blot. There was a gradually increased GRP78 expression from colorectal NNMs, carcinomas, to low-grade and high-grade adenomas (P<0.05), while up-regulated GRP94 expression from NNM, low-grade adenoma, high-grade adenoma, to carcinoma (P<0.05). The expression was similar in all the carcinoma cell lines. GRP78 expression was negatively correlated with lymphatic invasion or low GRP94 expression of the carcinomas (P<0.05), while there was no correlation of GRP94 expression with other parameters of carcinomas (P>0.05). Multivariate analysis showed that venous invasion, lymph node metastasis and UICC staging (P<0.05), but not age, sex, tumor size, differentiation, depth of invasion, lymphatic invasion, GRP78 and GRP94 expression (P>0.05), were independent prognostic factors for carcinomas. It is suggested that up-regulated expression of GRP78 and GRP94 could possibly be involved in the pathogenesis of colorectal carcinomas.

Inhibitor of growth 5, a tumor suppressor protein, can interact with p53, thereby inhibiting cell growth and inducing apoptosis. Inhibitor of growth 5 overexpression results in a reduction in colony-forming efficiency and cell population in S phase. To clarify the roles of inhibitor of growth 5 in tumorigenesis and progression of colorectal carcinomas, we examined inhibitor of growth 5 expression by immunohistochemistry on a tissue microarray containing colorectal carcinomas (n = 306), adenomas (n = 69), and nonneoplastic mucosa (n = 288) and compared this with clinicopathologic parameters of the carcinomas. In addition, inhibitor of growth 5 expression in colorectal carcinoma tissues and cell lines (DLD-1, HCT-15, SW480, and WiDr) was analyzed by Western blot and reverse transcriptase-polymerase chain reaction. It was found that the inhibitor of growth 5 protein was localized to the nuclei of colon carcinoma cells with no differences at mRNA levels. Among 18 frozen samples of colorectal carcinoma, significantly increased expression of inhibitor of growth 5 protein was observed in the carcinoma in comparison with adjacent mucosa in 14 cases (77.8%; P < .05), and 71.4% (10/14) of carcinoma cases exhibited up-regulated inhibitor of growth 5 mRNA expression. Decreased inhibitor of growth 5 expression was detected by immunohistochemistry in colorectal carcinoma, compared with non-neoplastic mucosa and adenoma (P < .05). Nuclear inhibitor of growth 5 expression was negatively correlated with tumor size, depth of invasion, degree of dedifferentiation, and Union Internationale Contre le Cancer staging (P < .05). In contrast, cytoplasmic inhibitor of growth 5 expression was positively correlated with depth of invasion, lymphatic invasion, and Union Internationale Contre le Cancer staging (P < .05). It was suggested that aberrant inhibitor of growth 5 expression may contribute to pathogenesis, growth, and invasion of colorectal carcinomas and could be considered as a promising marker to gauge aggressiveness of colorectal carcinomas., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Glycogen synthase kinase 3beta is reported to repress Wnt/beta-catenin pathway and regulate the balance between cellular proliferation and apoptosis. Its inactivation by ser(9) phosphorylation might play a critical role in the tumorigenesis and development of malignancies. Here, the expression of phosphorylated glycogen synthase kinase 3beta at ser(9) was examined in gastric carcinoma and adjacent non-neoplastic mucosa by immunohistochemistry and Western blot, and compared with the clinicopathological parameters of carcinomas, including the expression of vascular endothelial growth factor, extracellular matrix metalloproteinase inducer and beta-catenin, and microvessel density labeled by CD34, as well as survival data. Gastric carcinoma cell lines (MKN28, MKN45, AGS, GT-3 TKB, KATO-III and HGC-27) were studied for the phosphorylated kinase by Western blot and for its encoding mRNA by RT-PCR, followed by sequencing. All carcinoma cell lines showed strong expression of the phosphorylated kinase and its encoding mRNA.There were two isoforms of glycogen synthase kinase 3beta in all carcinoma cell lines and a synonymous mutation in HGC-27 carcinoma cell line at codon 65(GGA-->GGT: Gly). The phosphorylated kinase was localized in the cytoplasm of gastric carcinoma cell lines or carcinomas. It was more expressed in gastric carcinomas than that in non-neoplastic mucosa (P < .05) in line with the data of Western blot. There was a higher expression of the phosphorylated kinase in men carcinoma patients than that in women (P < .05). Its expression was positively correlated with depth of invasion, lymphatic and venous invasion, lymph node metastasis, Union Internationale le Contre Cancer staging, expression of vascular endothelial growth factor and extracellular matrix metalloproteinase inducer in gastric carcinoma (P < .05). Survival analysis indicated the phosphorylated kinase expression to be positively linked to poor prognosis (P< 05), but not independent (P>.05). Three independent prognostic factors, depth of invasion, lymphatic and venous invasion, concordantly influenced its relationship with prognosis (P < .05). Our study indicated that up-regulated expression of phosphorylated glycogen synthase kinase 3beta at ser(9) was closely linked to gastric carcinogenesis and subsequent progression, and could be employed as a good indicator of aggressive behaviors and prognosis of gastric carcinoma., (Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.)

Primary endometrioid adenocarcinoma rarely occurs in the vagina. Occasionally, endometrioid adenocarcinoma has a microglandular pattern. Herein, a case of primary endometrioid adenocarcinoma of the vagina with a microglandular pattern arising from pre-existing endometriosis long after a hysterectomy, is described. A 57-year-old postmenopausal woman developed a vaginal discharge over one decade after undergoing a hysterectomy. Microscopic examination of the vaginal smear and a biopsy specimen demonstrated an atypical glandular proliferation composed of columnar cells with occasional intracytoplasmic mucin and bland nuclear morphology, showing microcysts and numerous neutrophils within and around cysts. Immunohistochemically, the neoplastic cells were diffusely positive for CK7, MUC1, ER, and PR, and focally positive for vimentin, CEA, CK5/6, p63, p16(INK4a), and p53. A portion of residual endometrioid adenocarcinoma was identified adjacent to foci of endometriosis in the vaginectomy specimen. The patient has done well without evidence of recurrent disease for 1 year after surgery. Pathologists are encouraged to be aware of the occurrence of endometrioid adenocarcinoma associated with endometriosis in the vaginal stump after hysterectomy, and microglandular morphology which might be a source of misinterpretation.

Japan is a high-risk region for gastric carcinoma with a comparatively early stage and favorable prognosis. To clarify the pathobiological behaviors and prognosis of Japanese gastric adenocarcinoma, we analyzed the clinicopathological characteristics of different WHO subtypes of carcinomas. The expression of ki-67, CPP32, p53, FHIT, maspin, parafibromin, GRP78, GRP94, EMMPRIN, VEGF, P-GSK3beta-ser9, fascin, cortactin, Arp2, Arp3 MUC-2, MUC-5AC and MUC-6 was examined using immunohistochemistry and tissue microarrays. The majority of cases were well-, poorly-, or moderately-differentiated subtype, whereas the minority were papillary or signet ring cell carcinoma (SRC). Patients with poorly-differentiated or SRC carcinoma were predominantly young and female. Poorly-differentiated and mucinous carcinomas were larger, with deeper invasion, more venous or lymphatic invasion, frequent lymph node involvement and peritoneal dissemination, or higher staging. The SRC group exhibited weaker expression of ki-67, CPP32, p53, parafibromin, GRP78, GRP94, P-GSK3beta-ser9, VEGF or cortactin. The moderately-differentiated subtype exhibited lower expression of FHIT and Arp3 positivity. The poorly-differentiated group showed weaker expression of CPP32, EMMPRIN, MUC-2, MUC-5AC, and MUC-6. Survival analysis indicated that the patients with poorly-differentiated or mucinous subtypes had a lower cumulative survival rate than those with papillary, well-, moderately-differentiated, or SRC carcinomas (P<0.05). The age, invasive depth, lymphatic invasion, peritoneal dissemination, and WHO classification were independent prognostic factors for carcinoma patients (P<0.05). It was suggested that poorly-differentiated and mucinous subtypes are more aggressive and of unfavorable prognosis among Japanese gastric carcinomas. Lower levels of proliferation and apoptosis, as well as alterations in tumor suppressor genes, mucin production and ER stress protein played important roles in the pathogenesis of poorly-differentiated and SRC carcinomas.

To clarify the role of REG IV, a new member of the regenerating gene (REG) family, in tumorigenesis and progression of colorectal carcinoma (CRC), 320 CRC specimens, 123 corresponding adjacent non-cancerous mucosa (ANCMs), 46 corresponding non-adjacent non-cancerous mucosa (NANCMs) and 86 adenomas were investigated immunohistochemically to compare REG IV expression with clinicopathological features. In addition, double immunofluorescence labeling was performed to analyze the localization of REG IV and the intestinal mucin, MUC2. The expression of REG IV in CRCs was significantly lower than in NANCMs, ANCMs or adenomas, and inversely correlated with poor differentiation and venous invasion. In cases of ANCM, REG IV expression was positively correlated with the depth of invasion, lymph node metastasis and Duke's staging of corresponding cases. The expression of REG IV in CRC was significantly linked to that of MUC2 and the EGFR phosphorylated on Tyr1068, but not to that of MUC5AC, EGFR, Akt, or Akt phosphorylated on Ser473 or Thr308. The double immunofluorescence revealed coexpression, but independent localization, of REG IV and MUC2 in NANCMs, ANCMs, adenomas and CRCs, except for mucinous carcinomas. Univariate analysis using the Kaplan-Meier method indicated no correlation between REG IV expression and the cumulative survival rate of CRC patients. In conclusion, REG IV expression was upregulated in ANCMs and adenomas, then decreased in CRCs. This indicated that REG IV overexpression may be an early event in CRC carcinogenesis. Its expression in CRCs was positively linked to MUC2 and phosphorylation of the EGFR on Tyr1068, suggesting that REG IV may be a useful marker for intestinal type mucinous carcinoma and a good candidate as a molecular therapeutic target for CRCs.

Although the biologic function of Reg IV is poorly understood, it has been reported that Reg IV is a potent activator of the epidermal growth factor receptor/Akt/AP-1 signaling pathway in colon cancer cells and closely linked with the inhibition of apoptosis. To clarify the role of Reg IV in gastric carcinogenesis and subsequent progression, we examined its expression by immunohistochemistry and in situ hybridization on tissue microarray containing gastric carcinoma, adjacent nonneoplastic mucosa, adenoma, intestinal metaplasia, or gastritis. Gastric carcinoma cell lines (MKN28, AGS, MKN45, KATO-III, and HGC-27) were studied for Reg IV expression by Western blot and reverse transcriptase-polymerase chain reaction followed by sequencing. Frozen samples of gastric carcinoma and adjacent nonneoplastic mucosa were subjected to Western blot, and patient serum, to enzyme-linked immunosorbent assay for Reg IV. Gastric carcinoma cell lines showed different levels of Reg IV mRNA and its encoding protein. The Reg IV protein expression was gradually decreased from intestinal metaplasia, adenoma, and carcinoma to gastritis (P < .05). The positive rate of its mRNA was higher in intestinal metaplasia than carcinoma or nonneoplastic mucosa (P < .05). Elevated serum Reg IV level in gastric carcinoma patients was detected in comparison with that in health individuals (P < .05). Reg IV expression was significantly correlated with the MUC-2 and MUC-5AC expression (P < .05). Among histologic subtypes of the World Health Organization, signet ring cell carcinoma more frequently expressed Reg IV than the others (P < .05), whereas it is the converse for the poorly differentiated group (P < .05). Our study indicated that Reg IV expression experienced up-regulation in gastric intestinal metaplasia and adenoma and then down-regulation with malignant transformation of gastric epithelial cells. It was suggested that Reg IV expression should be considered as a good biomarker for gastric precancerous lesions and was especially related to the histogenic pathway of signet ring cell carcinoma.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is a phosphatase that antagonizes the phosphoinositol-3-kinase/AKT signaling pathway and suppresses cell survival as well as cell proliferation. To investigate the molecular role of PTEN expression and mutation in colorectal carcinomas, we performed immunohistochemistry to detect PTEN expression on tissue microarray containing colorectal carcinomas and corresponding adjacent non-cancerous mucosa. PTEN mutation was studied from exon 1 to 9 by PCR, followed by direct sequencing. PTEN expression was then compared with clinicopathological parameters and prognosis of the tumor, including caspase-3 expression. In the present study, PTEN expression was stronger in the adjacent non-cancerous mucosa than carcinoma (P<0.001). Low PTEN expression was positively correlated with tumor size, depth of invasion, lymphatic invasion, lymph node metastasis, higher Dukes staging and reduced caspase-3 expression (P<0.05), but not with venous invasion or differentiation (P>0.05). Univariate analysis suggested that the patients without PTEN expression had shorter survival than the patients with its expression (P=0.003). Multivariate analysis indicated that lymphatic invasion, venous invasion, and PTEN expression were independent prognostic factors for overall colorectal carcinomas (P<0.05). The analysis of mutations revealed only one synonymous mutation in exon 8 (codon 312 Asp: GACright curved arrow GAT). These results suggested that down-regulated PTEN expression was involved in the pathogenesis, invasion and metastasis of colorectal carcinomas possibly by regulating the balance between apoptosis and proliferation. PTEN expression may be a good marker for the prognosis of colorectal carcinoma.

Maspin, a serine protease inhibitor related to the serpin family, can inhibit invasion and metastasis of malignancies although direct evidence of the clinicopathologic significance of cytoplasmic relative to nuclear expression is limited. Here, maspin expression was examined on tissue microarrays containing lung carcinoma (n=155) and adjacent noncancerous tissue (n=20) and also 4 lung carcinoma cell lines (LC-1/Sq, LC-IF, PC-14, and AoI) by immunohistochemistry. Maspin expression was compared with clinicopathologic parameters of the tumors. Maspin expression showed positive nuclear staining in basal cells, LC-IF, and PC-14 cell lines, and also cytoplasmic immunoreactivity in secretory and ciliated cells, LC-1/Sq cell line. Cytoplasmic staining was the lowest in adenocarcinoma (AD) and the highest in squamous cell carcinoma as compared with other types of lung carcinoma (P<0.05), and positively correlated with expression of p53 and caspase-3 (P<0.05). The cytoplasmic one showed stronger immunoreactivity in male carcinoma patients than female ones (P<0.05). The nuclear maspin expression gradually increased through squamous cell carcinoma, AD, large cell carcinoma to small cell carcinoma (P<0.05) and was also positively associated with the levels of vascular epithelial growth factor and extracellular matrix metalloproteinase inducer expression (P<0.05). Kaplan-Meier analysis indicated that the cytoplasmic or nuclear maspin expression was not a good prognostic marker for lung carcinomas overall (P>0.05), but the cytoplasmic pattern pointed to good survival for AD cases (P<0.05). It was concluded that the cytoplasmic and nuclear expression patterns of maspin are involved in the cellular differentiation of normal lung tissue and the histogenesis of different lung carcinomas. The cytoplasmic maspin may play an important role in lung carcinomas by regulating apoptosis and thus is a favorable prognostic marker for AD patients, whereas the nuclear location may be linked to promotion of angiogenesis.

Background: Tumor metastasis depends on cell adhesion, motility and deformability, resulting from quantitative alterations and rearrangement of actin-related protein (Arp) 2 and 3. The aim of this study was to clarify the roles of both molecules in tumorigenesis and progression of gastric carcinomas., Patients and Methods: Immunohistochemistry (IHC) was employed on tissue microarray containing gastric carcinomas, adjacent metaplasia and gastritis using antibodies against Arp2 and Arp3 with a comparison of their expression with clinicopathological parameters of carcinomas. Gastric carcinoma cell lines (MKN28, AGS, MKN45, KATO-III and HGC-27) were studied for Arp2 and Arp3 protein by IHC., Results: Both proteins were expressed at low levels in gastritis compared with carcinomas (p < 0.05). Arp2 was more frequently expressed in intestinal metaplasia than in carcinoma and gastritis (p < 0.05). Most gastric carcinoma cell lines showed expression at different levels. Expression was positively correlated with tumor size, depth of invasion, venous invasion, Union Internationale Contre le Cancer (UICC) staging and expression of cortactin or fascin (p < 0.05), but not with age, sex, lymphatic invasion or lymph node metastasis (p > 0.05). There was stronger positivity of Arp3 in intestinal- than diffuse-type carcinomas (p < 0.05). A positive relationship between Arp2 and Arp3 proteins was noted (p < 0.05). Univariate analysis indicated that the cumulative survival rate of patients with positive Arp2 or Arp3 expression was not different from those without their expression (p > 0.05). Multivariate analysis showed that age, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren's classification were independent prognostic factors for carcinomas (p < 0.05)., Conclusion: Aberrant expression of Arp2 and Arp3 is possibly involved in pathogenesis, growth, invasion and progression of gastric carcinomas. Distinct Arp3 expression underlies the molecular mechanisms for the differentiation of intestinal- and diffuse-type carcinomas. They were considered as objective and effective markers to indicate the pathobiological behaviors of gastric carcinomas.

Purpose: Galectin-3, a beta-galactoside-binding animal lectin is a multifunctional protein, which regulates cell growth, cell adhesion, cell proliferation, angiogenesis, and apoptosis, and in turn contributes to tumorigenesis and metastasis. The aim of this study was to clarify the role or related mechanisms of galectin-3 in lung carcinogenesis., Methods: We administrated 4-(methylnitrosamino)-1-(3-pyridyle)-1-butanone (NNK), a powerful chemical carcinogen into galectin-3 wild-type (gal3+/+) and galectin-3 knock-out (gal3-/-) CD1 mice by intraperitoneal injection, examined the expression status of 22,690 mouse genes of the NNK-induced tumors using Affymetrix GeneChip mouse expression 430 A arrays, and then analyzed functional network and gene ontology by Ingenuity Pathway Analysis. Real-time PCR was also employed to partially confirm the genechip data., Results: Compared with the gal3+/+ mice, the incidence of lung tumors was significantly low in gal3-/- mice after 32 weeks (28.6 vs 52.1%, P < 0.05). Pathway analysis indicated that galectin-3 up-regulated carcinogenesis-related genes (e.g. B-cell receptor, ERK/MAPK, and PPAR signalings) in normal condition, and lung cancer and NNK-induced gene expression associated with cellular growth (e.g. Wnt/beta-catenin signaling) or immunological disease (e.g. EGF and PDGF signalings) in lung carcinogenesis with or without the galectin-3 control, respectively., Conclusion: Disrupted galectin-3 may attenuate the lung carcinogenesis due to its regulatory role in the B-cell receptor, ERK/MAPK, and PPAR signal pathways.

Glucose-related proteins (GRPs) are ubiquitously expressed in endoplasmic reticulum and able to assist in protein folding and assembly; consequently, they are considered as molecular chaperones. GRP78 and GRP94 expression was induced by glucose starvation and up-regulated in the malignancies. To clarify the roles of both molecules in tumorigenesis and progression of gastric carcinomas, immunohistochemistry was used on tissue microarray containing gastric carcinomas, adenomas, and nonneoplastic mucosa using the antibodies against GRP78 and GRP94, with a comparison of their expression with clinicopathological parameters of carcinomas. Gastric carcinoma cell lines (MKN28, AGS, MKN45, KATO-III, and HGC-27) were studied for both proteins by immunohistochemistry and Western blot. There was more expression of both proteins in gastric carcinoma and adenoma than in nonneoplastic mucosas (P < .05). All gastric carcinoma cell lines showed their expression at different levels. They were positively correlated with tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis, and Union Internationale Contre le Cancer staging (P < .05), with positive relationship between both proteins (P < .05). Univariate analysis indicated the postsurgical cumulative survival rate of patients with positive GRP78 or GRP94 expression to be lower than that in those without GRP78 or GRP94 expression (P < .05), but the close link disappeared if stratified according to depth of invasion (P > .05). Multivariate analysis showed that age, depth of invasion, lymphatic invasion, lymph node metastasis, Union Internationale Contre le Cancer staging, and Lauren classification (P < .05), but not GRP78 and GRP94 expression, were independent prognostic factors for carcinomas (P > .05). Up-regulated expression of GRP78 and GRP94 was possibly involved in pathogenesis, growth, invasion, and metastasis of gastric carcinomas. They were considered objective and effective markers for the aggressive behavior and poor prognosis in gastric carcinomas.

Purpose: Pim-3, a member of the proto-oncogene Pim family with serine/threonine kinase activity was aberrantly expressed in cancerous lesions of endoderm-derived organs such as liver, pancreas, and colon. The aim of this study was to clarify the role of Pim-3 expression in the tumorigenesis and the development of gastric carcinomas., Methods: Pim-3 expression was immunohistochemically examined on the tissue microarrays containing primary (n = 285) and metastastic (n = 37) sites of gastric carcinomas, in comparison with adenoma (n = 48) and non-cancerous mucosa (n = 84). It was also compared with the clinicopathological parameters of gastric carcinomas., Results: Pim-3 expression was enhanced in adenoma (64.6%) and metastasis sites of gastric carcinoma (73.0%), to a lesser degree in primary sites of gastric carcinoma (39.3%) when compared to non-cancerous mucosa (13.1%, p < 0.0001). Pim-3 expression levels were higher in intestinal-type than diffuse-type gastric carcinoma (p = 0.018). Pim-3 expression was closely correlated with sex (p = 0.047), lymphatic (p = 0.019) and venous invasion (p = 0.014). Pim-3 expression was correlated significantly with vascular endothelial growth factor (VEGF, p = 0.009) and extracellular matrix metalloproteinase inducer (EMMPRIN, p = 0.032), both of which are presumed to be involved in neovascularization, a crucial step for metastasis. On the contrary, phosphatase and tensin homology deleted from human chromosome 10 (Pten) negative gastric carcinomas exhibited higher Pim-3 expression than Pten positive ones (p = 0.042). There was no relationship between Pim-3 expression and MVD in gastric carcinomas (p = 0.715). Furthermore, patients with Pim-3 positive gastric cancer, showed a lower cumulative survival rate than those with Pim-3 negative gastric cancer (p = 0.014) and Pim-3 positive was also identified as an independent prognostic factor for gastric carcinoma patients (p = 0.006)., Conclusions: Aberrant Pim-3 expression was involved in gastric adenoma-adenocarcinoma sequence and subsequent invasion and metastasis process in gastric cancer. Moreover, Pim-3 may be employed to predict the prognosis of gastric cancer patients. Distinct Pim-3 expression underlies the molecular mechanisms for the differentiation of intestinal-type and diffuse-type carcinomas.

Parafibromin is a protein encoded by the hyperparathyroidism 2 oncosuppressor gene and its downregulated expression is involved in pathogenesis of parathyroid carcinomas. To clarify the roles of parafibromin expression in tumourigenesis and progression of gastric carcinomas, it was examined by immunohistochemistry (IHC) on tissue microarray containing gastric carcinomas (n = 508), adenomas (n = 45) and gastritis (n = 49) with a comparison of its expression with clinicopathological parametres of carcinomas. Gastric carcinoma cell lines (MKN28, AGS, MKN45, KATO-III and HGC-27) were studied for parafibromin expression by IHC and western blot. Parafibromin expression was localised in the nucleus of gastric epithelial cells, adenoma, carcinoma cells and cell lines. Its expression was gradually decreased from gastritis to gastric carcinoma, through gastric adenomas (p < 0.05) and inversely correlated with tumour size, depth of invasion, lymphatic invasion, lymph node metastasis and Union Internationale Contre le Cancer (UICC) staging (p < 0.05) but not with sex or venous invasion (p > 0.05). Parafibromin was strongly expressed in older carcinoma patients compared with younger ones (p < 0.05). There was stronger positivity of parafibromin in intestinal-type than diffuse-type carcinomas (p < 0.05). Univariate analysis indicated cumulative survival rate of patients with positive parafibromin expression to be higher than without its expression (p < 0.05). Multivariate analysis showed that age, tumour size, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren's classification but not sex, venous invasion or parafibromin expression were independent prognostic factors for carcinomas(p < 0.05). Downregulated parafibromin expression possibly contributed to pathogenesis, growth, invasion and metastasis of gastric carcinomas. It was considered as a promising marker to indicate the aggressive behaviours and prognosis of gastric carcinomas.

Maspin, a serine protease inhibitor related to the serpin family, can suppress invasion and metastasis of malignancies. To clarify the role of maspin in the genesis and progression of gastric carcinomas, its expression pattern and level were studied by immunohistochemistry on tissue microarrays containing gastric carcinoma (n = 237), normal gastric mucosa (n = 23), intestinal metaplasia (n = 38), and adenoma (n = 42); and the findings were compared with clinicopathological parameters. Furthermore, maspin expression in the gastric carcinoma cell lines (HCG-27, MKN28, and MKN45) was examined by immunohistochemistry and Western blotting. We found that cytoplasmic and nuclear maspin expression paralleled each other (P < .05) and decreased from intestinal metaplasia, adenoma, and carcinoma to normal gastric mucosa (P < .05). A significant positive association was noted with depth of invasion, lymphatic invasion, lymph node metastasis, and TNM stage (P < .05) but not with sex or Lauren's classification (P > .05). Univariate and multivariate analyses indicated that expression of maspin correlated negatively with cumulative patient survival in gastric carcinoma (P < .05) but was not an independent factor in the prognosis. The 2 independent factors, depth of invasion and lymphatic invasion, influenced the relation between nuclear maspin expression and survival, whereas only depth of invasion correlated with cytoplasmic maspin. Our study indicated that maspin expression experiences upregulation in gastric precancerous lesions and then slight downregulation with malignant transformation. High expression may paradoxically promote invasion and metastasis of gastric carcinomas and could be considered a good marker for the pathobiological behaviors of gastric carcinomas.

Background: Maspin, a serine protease inhibitor related to the serpin family, can inhibit invasion and metastasis of malignancies. This study aimed to explore the roles of maspin expression in the tumorigenesis and progression of colorectal adenocarcinoma (CRA)., Materials and Methods: Maspin expression was examined on tissue microarrays containing CRAs (n = 119), adjacent adenoma (n = 22), adjacent non-cancerous mucosa (n = 118) and metastases (n = 67) by immunostaining. Microvessel density (MVD) was determined after labeling with anti-CD34 antibody using immunnostaining. The maspin expression was compared with clinicopathological parameters of the tumors, including expression of p53, Ki-67 and tenascin, MVD, and survival data., Results: Maspin expression showed significant increase from colorectal non-cancerous mucosa to adenocarcinoma through adenoma (p < 0.05). Maspin expression correlated negatively with liver metastasis of CRA (p < 0.05), positively with tenascin expression (p < 0.05), but not with tumor size, depth of invasion, local invasion via vessels, lymph node metastasis, differentiation, expression of Ki-67 and p53 or MVD (p > 0.05). Maspin expression in the metastases of CRA was significantly consistent with their corresponding primary foci (p < 0.05). Kaplan-Meier analysis revealed no significant relationship between maspin expression and survival time of carcinoma patients (p > 0.05)., Conclusion: Up-regulated maspin expression was involved in colorectal adenoma-adenocarcinoma sequence. Low maspin expression is closely linked to the liver metastasis of CRA possibly through degradation of the extracellular matrix-tenascin to enhance carcinoma cell mobility.

Aim: To seek good markers to predict invasion and metastasis of gastrointestinal adenocarcinoma (GIA)., Methods: Expression of KAI1 and tenascin were examined on tissue microarrays containing gastric adenocarcinoma (n = 98), colorectal adenocarcinoma (n = 125), gastric adjacent non-cancerous mucosa (n = 95) and colorectal adjacent non-cancerous mucosa (n = 112) by immunostaining. Microvessel density (MVD) in GIA was labelled using anti-CD34 antibody by immunostaining. Expression of KAI1 and tenascin, and MVD were compared with clinicopathological features of tumours, including PTEN (phosphatase and tensin homology deleted from human chromosome 10) and EMMPRIN (extracellular matrix metalloproteinase inducer) expression., Results: KAI1 expression was higher in GIAs than in their adjacent non-cancerous mucosa (p<0.05). KAI1 and tenascin expression showed a significantly negative association with liver metastasis of GIA (p<0.05), but not with depth of invasion, venous invasion or lymph node metastasis (p>0.05). A significantly negative relationship was observed between EMMPRIN and tenascin expression in GIA (p<0.05). MVD was positively correlated with depth of invasion, venous invasion, lymph node metastasis and liver metastasis of tumours (p<0.05), whereas it was negatively correlated with PTEN expression (p<0.05)., Conclusions: Up-regulated KAI1 expression may play an important part in malignant transformation of gastrointestinal epithelial cells. Reduced expression of KAI1 and tenascin might underlie the molecular basis of liver metastasis of GIA. Angiogenesis is a key event in the invasion and metastasis of GIA. These markers might be used to indicate liver metastasis of GIA in clinicopathological practice.

Ki-67 immunostaining is commonly used for assessing cell proliferation, but studies of its use as a prognostic indicator have revealed discordant results in gastric cancer patients. Recently, antibodies for phosphorylated histone H3 have been used to identify dividing cells because of its precise overexpression in mitosis. The authors tested the hypothesis that phosphorylated histone H3 overexpression might be a good prognostic indicator for gastric cancer patients by conducting an immunohistochemical comparison with Ki-67 in gastric cancer samples. One hundred twenty-two surgically resected primary cases were selected and histologically categorized in accordance with Lauren's classification. No correlation was found between phosphorylated histone H3 and Ki-67 regarding overexpression. However, correlations between phosphorylated histone H3 overexpression and clinicopathologic variables were noted for histologic type (intestinal type predominant in high labeling indices [LIs], defined as over the value of the 75th percentile; P<0.01), vessel invasion (positive in high LIs; P=0.05), and lymph node metastasis (positive in high LIs; P=0.04). With regard to Ki-67 overexpression, no correlation was evident with the clinicopathologic variables except histologic type (intestinal type predominant; P=0.05). By the Kaplan-Meier method with the log-rank test, cases overexpressing phosphorylated histone H3 showed a poorer prognosis than cases with low expression (P<0.01). In contrast, Ki-67 expression did not influence prognosis. Multivariate analyses indicated phosphorylated histone H3 overexpression to be an independent prognostic factor, together with lymphatic invasion and venous invasion (P<0.01). In conclusion, it seems likely that phosphorylated histone H3 plays an important role in the prognosis of gastric cancer, and its immunohistochemical investigation is useful for the prediction of prognosis in gastric cancer.

Background: Gastric carcinoma is still a major leading cause of cancer death in East Asia. Since angiogenesis is a necessary condition for invasion and metastasis, its regulation is of essential significance., Materials and Methods: Expressions of MMP-2, MMP-9 and VEGF were examined with microarray of gastric carcinoma tissue samples (n = 249) by immunostaining. In addition, microvessel density (MVD) was assessed after labelling with the anti-CD34 antibody. Data were cross-compared with clinicopathological parameters of tumors, including PTEN expression., Results: Expressions of MMP-2, MMP-9 and VEGF were positively correlated with tumour size, depth of invasion, lymphatic and venous invasion, lymph node metastasis, UICC staging and MVD of gastric carcinomas (p < 0.05).VEGF expression was positively linked with levels of MMP-2 and MMP-9 (p < 0.05), but negatively with PTEN (p < 0.05). The latter was also inversely associated with the MVD in gastric carcinomas (p < 0.05)., Conclusion: MMP-2, MMP-9 and VEGF largely contribute to the angiogenesis and progression of gastric carcinomas. PTEN might inhibit the processes by down-regulating VEGF expression. These parameters should be regarded as good markers to indicate pathobiological behaviours of gastric carcinomas.

Background: Gastrointestinal carcinogenesis generally follows the adenoma-adenocarcinoma sequence and tumor metastasis determines the survival time of the patients. The expressions of p53 and ki-67 in gastrointestinal adenoma and adenocarcinoma (GIA) were explored and their clinicopathological significance determined., Materials and Methods: The expressions of mutant p53 and ki-67 were examined on tissue microarray containing GIA, adjacent mucosa or adenoma and metastases by immunostaining. Their expressions were compared with the clinicopathological parameters of tumors., Results: The expressions of mutant p53 and ki-67 were gradually increased from gastrointestinal mucosa to adenocarcinoma through adenoma (p<0.05). Mutant p53 expression showed a positive association with depth of invasion, local invasion via vessels and lymph node metastasis of GIA (p<0.05). Ki-67 expression was positively correlated with local invasion via vessels and negatively with dedifferentiation and liver metastasis of GIA (p<0.05). The expressions of both markers in metastases of GIA were consistent with their corresponding primary foci (p < 0.05). A positive association between both markers was found in the primary foci of GIA (p<0.05)., Conclusion: The up-regulated expressions of mutant p53 and ki-67 are involved in the carcinogenesis and progression of GIA. They appear to be objective and effective markers to reflect the development of GIA.

JC virus (JCV), a ubiquitous polyoma virus that commonly infects humans, was first identified as the etiologic agent for the fetal demyelinating disease, progressive multifocal leukoencephalopathy. Recently, a number of reports have documented detection of JCV in samples derived from several types of neural as well as non-neural human tumors. It has been suggested that oncogenicity of JCV depends on a T antigen having a strict structural homology to the T antigen of simian virus 40. To clarify whether JCV might have a potential role with regard to colorectal cancers, we investigated the presence of its genome in a series of cases along with colorectal adenomas and normal colonic mucosa, targeting T antigen, VP and agnoprotein by nested polymerase chain reaction and Southern blotting and T antigen by immunohistochemistry. While VP and agnoprotein were not found in any of the samples examined, T antigen was detected in 6 of 23 colorectal cancers (26.1%) and 1 of 21 adenomas (4.8%), but none of 20 samples of normal colonic mucosa. No clear and diffuse staining with anti-T-antigen antibodies (1:100) could be detected, and there was no correlation with CD20-positive cells, which might have indicated JCV latent infection of B lymphocytes. Presence of T antigen did not influence clinicopathological variables, including survival. In one colonic cancer case positive for T antigen together with lymph node metastasis, DNA extracted from cancer cells in the lymph node revealed no detection of T antigen. Our results are in the intermediate position between the high T antigen rate (81%) in one report and the lack of it (0%) in another focused on colon cancers. It was concluded that T antigen might be integrated in cancer cells in approximately one fourth of Japanese colon cancer cases without clear and diffuse expression of the protein, suggesting a possible role in oncogenesis which might involve a hit-and-run mechanism.

Ovarian clear cell adenocarcinoma (CCC) is the second most common subtype of ovarian cancer after high-grade serous adenocarcinomas. CCC tends to develop resistance to the standard platinum-based chemotherapy, and has a poor prognosis when diagnosed in advanced stages. The ANXA4 gene, along with its product, a Ca++-binding annexin A4 (ANXA4) protein, has been identified as the CCC signature gene. We reported two subtypes of ANXA4 with different isoelectric points (IEPs) that are upregulated in CCC cell lines. Although several in vitro investigations have shown ANXA4 to be involved in cancer cell proliferation, chemoresistance, and migration, these studies were generally based on its overexpression in cells other than CCC. To elucidate the function of the ANXA4 in CCC cells, we established CCC cell lines whose ANXA4 expressions are stably knocked down. Two parental cells were used: OVTOKO contains almost exclusively an acidic subtype of ANXA4, and OVISE contains predominantly a basic subtype but also a detectable acidic subtype. ANXA4 knockdown (KO) resulted in significant growth retardation and greater sensitivity to carboplatin in OVTOKO cells. ANXA4-KO caused significant loss of migration and invasion capability in OVISE cells, but this effect was not seen in OVTOKO cells. We failed to find the cause of the different IEPs of ANXA4, but confirmed that the two subtypes are found in clinical CCC samples in ratios that vary by patient. Further investigation to clarify the mechanism that produces the subtypes is needed to clarify the function of ANXA4 in CCC, and might allow stratification and improved treatment strategies for patients with CCC. [ABSTRACT FROM AUTHOR]