Overexpression of annexin 1 in pancreatic cancer and its clinical significance

AIM: To investigate the expression of annexin I in pancreatic cancer and its relationship with the clinicopathologic factors,and to evaluate its potential clinical significance.METHODS: Annexin I expression was analyzed by Western blot and immunohistochemical staining in pancreatic adenocarcinoma and multi-tissue microarrays (MTAs).RESULTS: Western blot analysis showed that annexin I was overexpressed in 84.6% (11/13) pancreatic ductal adenocarcinomas. Immunohistochemistry analysis of pancreatic cancer in MTAs showed that annexin I protein was 71.4%(30/42) positive which was markedly increased compared with that in the tumor matched normal pancreas tissues 18.4%(7/38) (P<0.01). In the meantime, the high expression of annexin 1 was correlated with the poor differentiation of pancreatic adenocarcinoma.CONCLUSION: Annexin 1 overexpression is a frequent biological marker and correlates with the differentiation of pancreatic cancer during tumorigenesis.

Profiling of differentially expressed genes in human Gastric carcinoma by cDNA expression array

AIM:To investigate the expression of cancer related genes in gastric cacinoma(GC)through the use of Atlas-Human Cancer Array membranes with 588 well-characterized human genes related to cancer and tumor biology.METHODS:Hyb ridization of cDNA blotting membrane was performed with ^32P-labeled cDNA probes synthesized fromRNAisolated from gastric carcinoma and adjacent noncancerous gastric epithelial tissue.AtlasImage,which is a software specific to array,wasused to analyze the result.RESULTS:The differentially expression cell cycle/growth regulator in GC showed a stronger tendency toward cell proliferation with2.7-fold up-regulation of CK1.The promoter genes of apoptosis were down-regulated,including caspase-8 precursor,caspase-9and caspase-10.Among the oncogene/tumor suppressor genes.ABL2 was down-regulated.In addition.some genes were up-regulated,including matrix metalloproteinse 2(MMP-2),MMP-16(MT3-MMP),SKY,CD9 and semaphorinV.Anumber of genes were down-regulated,including neruroendocrine-dlg(NE-dlg),retinoic acid receptor gamma and tumor suppressor DCC colorectal.In general.The expression of the cancer progression genes were up-regulated.while the expression of anti-cancer progression genes were down-regulated.CONCLUSION:Investigation of these genes should help to disclose the molecular mechanism of the onset,progression and prognosis of GC.Several genes are reported herein to be altered in GCfor the first time,The quick and high-throughout methodof profiling gene expression by cDNA array provides us with an overview of key factors that may inolved in GC,and may aid the study of GC carcinogenesis and provide molecular targets for diagnosis and therapy.The precise relationship between the altered genes and gastric carcinogenesis is a matter for further investigation.

Loss of clusterin both in serum and tissue correlates with the tumorigenesis of esophageal squamous cell carcinoma via proteomics approaches

AIM: To identify the differentially secreted proteins or polypeptides associated with tumorigenesis of esophageal squamous cell carcinoma (ESCC) from serum and to find potential tumor secreted biomarkers.METHODS: Proteins from human ESCC tissue and its matched adjacent normal tissue; pre-surgery and postsurgery serum; and pre-surgery and normal control serum were separated by two-dimensional electrophoresis (2-DE)to identify differentially expressed proteins. The silverstained 2-DE were scanned with digital ImageScanner and analyzed with ImageMaster 2D Elite 3.10 software. A cluster of protein spots differentially expressed were selected and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). One of the differentially expressed proteins, clusterin, was downregulated in cancer tissue and pre-surgery serum, but it was reversed in post-surgery serum. The results were confirmed by semi-quantitative reverse-transcription (RT)-PCR and western blot.RESULTS: Comparisons of the protein spots identified on the 2-DE maps from human matched sera showed that some proteins were differentially expressed, with most of them showing no differences in composition, shape or density.Being analyzed by MALDI-TOF-MS and database searching,clusterin was differentially expressed and down-regulated in both cancer tissue and pre-surgery serum compared with their counterparts. The results were also validated by RTPCR and western blot.CONCLUSION: The differentially expressed clusterin may play a key role during tumorigenesis of ESCC. The 2DE-MS based proteomic approach is one of the powerful tools for discovery of secreted markers from peripheral.

Loss of myeloid-related proteins 8 and myeloid-related proteins 14 expression in human esophageal squamous cell carcinoma correlates with poor differentiation

AIM:To study the expression of myeloid-related proteins (MRP)8 and myeloid-related proteins(MRP)14 in human esophageal squamous cell carcinoma and to investigate if there was any correlation between MRP8 and MRP14 expression level and histopathological grade in these tumors. METHODS:In this study,65 cases of advanced esophageal squamous cell carcinoma were assessed for MRP8 and MRP14 expression using immunohistochemistry.Statistical analysis was performed for the comparison of MRP8 and MRP14 expression in normal and tumor tissues,and their relationship with clinicopathological features. RESULTS:Reduced or absent expression of MRP8 and MRP14 was observed in esophageal squamous cell carcinoma,with a significant difference between tumor tissues and normal tissues (P<0.01 and P<0.01 for MRP8 and MRP14,respectively). Poorly differentiated tumors presented a greater decrease than well and moderately differentiated tumors,with a correlation between their protein level and histopathological grading (P<0.001 and P<0.001,respectively).However,no significant association was found between MRP8 and MRP14 expression and age or gender (P>0.05). CONCLUSION:These findings suggest that the decreased expression of MRP8 and MRP14 might play an important role in the pathogenesis of human esophageal squamous cell carcinoma,being particularly associated with poor differentiation of tumor cells.

Downregulation of retinoic acid receptor-β_z expression is linked to aberrant methylation in esophageal squamous cell carcinoma cell lines

AIM:To study the role of hypermethylation in the loss of retinoic acid receptor β2(RARβ2) in esophageal squamous cell carcinoma (ESCC).METHODS:The role of hypermethylation in RARβ2 gene silencing in 6 ESCC cell lines was determined by methylation-specific PCR (MSP),and its methylation status was compared with RARβ2 mRNA expression by RT-PCR.The MSP results were confirmed by bisulfite sequencing of RARβ2promoter regions.RESULTS:Methylation was detected in 4 of the 6 cell lines,and the expression of RARβ2 was markedly downregulated in 3 of the 4 methylated cell lines. The expression of RARβ2 was restored in one RARβ2-downregulated cell line with the partial demethylation of promoter region of RARβ2 after 5-aza-2′-deoxycytidine (5-aza-dc) treatment.CONCLUSION:The methylation of the 5′ region may play an important role in the downregulation of RARβ2 in some ESCC cell lines, suggesting that multiple mechanisms contribute to the loss of RARβ2expression in ESCC cell lines.This study may have clinical applications for treatment and prevention of ESCC.

Expression of MRP14 gene is frequently down-regulated in Chinese human esophageal cancer

Migration inhibitory factor-related protein 14 (MRP 14) is one of calcium-binding proteins,referred as S 100A9. The heterodimeric molecule formed by MRP 14 with its partner MRP8 (S 100A8) is the major fatty acid carrier in neutrophils.The MRP8/14 complex has been also implicated in the intracellular transport of arachidonic acid and its precursors in keratinocytes. We show here the involvement of MRP14 in human esophageal cancer. In an initial study,mRNA differential display - reverse transcription polymerase chain reaction (DD-PCR) was performed with two esophageal carcinomas,one esophageal adenocarcinoma and matched normal adjacent mucosa. DD-PCR with the arbitrary primer OPA3 showed that one cDNA band was highly expressed in normal tissues,but disappeared or substantially decreased in tumor counterparts. It was later identified to be the 3'-end of migration inhibitory factor-related protein 14 (MRP14).Northern blotting,RT-PCR and Western blotting corroborated the down-regulation of MRP14 in 58/64 squamous cell carcinomas and 2/2 adenocarcinomas as compared with adjacent normal epithelia of the esophagus. MRP14 was undetectable in 3/3 esophageal-carcinoma cell lines. Immunochemistry demonstrated that expression of MRP14 was restricted to normal esophageal epithelia. No mutation was found in the genomic DNA of the MRP14 gene by PCR and directed DNA sequencing. Our finding suggested that the reduction of MRP14 expression is a frequent event in Chinese human esophageal cancer.

Gene expression profiles at different stages of human esophageal squamous cell carcinoma

AIM: To characterize the gene expression profiles in differentstages of carcinogenesis of esophageal epithelium.METHODS: A microarray containing 588 cancer relatedgenes was employed to study the gene expression profileat different stages of esophageal squamous cell carcinomaincluding basal cell hyperplasia, high-grade dysplasia,carcinoma in situ, early and late cancer. Principle componentanalysis was performed to search the genes which wereimportant in carcinogenesis.RESULTS: More than 100 genes were up or down regulatedin esophageal epithelial cells during the stages of basal cellhyperplasia, high-grade dysplasia, carcinoma in situ, earlyand late cancer. Principle component analysis identified aset of genes which may play important roles in the tumordevelopment. Comparison of expression profiles betweenthese stages showed that some genes, such as P160ROCK,JNK2, were activated and may play an important role inearly stages of carcinogenesis. CONCLUSION: These findings provided an esophagealcancer-specific and stage-specific expression profiles,showing that complex alterations of gene expression underliethe development of malignant phenotype of esophagealcancer cells.

Alterations in expression,proteolysis and intracellular localizations of clusterin in esophageal squamous cell carcinoma

AIM: To investigate biogenesis and intracellular Iocalizations of clusterin to elucidate the potential molecular mechanisms implicated in tumorigenesis of esophageal mucosa.METHODS: Semi-quantitative RT-PCR for multi-region alteration analysis, Western blot for different transcriptional forms and immunohistochemical staining for intracellular Iocalizations of clusterin were carried out in both tissues and cell lines of ESCC.RESULTS: The N-terminal deletions of the clusterin gene and the appearance of a 50-53 ku nuclear clusterin, an uncleaved, nonglycosylated, and disulfide-linked isoform,were the major alterations in cancer cells of esophagus.Naturally the 40 ku clusterin was located in the connective tissue of the lamina propria of epithelial mucosa and right under the basal membrane of epithelia, but it was disappeared in stromal mucosa of esophagus and the pre-matured clusterin was found positive in cancerous epithelia.CONCLUSION: The N-terminal deletion of clusterin may be essential for its alterations of biogenesis in ESCC.

Transglutaminase 3 expression in C57BL/6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornifiedenvelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then wedetermined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. Wefound a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cellsfrom E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed inthe granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin develop-ment provided several morphological evidences for the epidermal differentiation. The above findings suggest that theexpression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.

Expression patterns of esophageal cancer deregulated genes in C57BL/6J mouse embryogenesis

AIM:To investigate the expression patterns of esophageal squamous cell cancer deregulated genes in mid to late stages of C57BL/6J mouse embryogenesis,and the correlation between these genes in embryonic development and tumorigenesis of esophageal squamous cell cancer. METHODS:Reverse northern screening was performed to examine the expression patterns of esophageal cancer deregulated genes in C57BL/6J mouse embryogenesis.To confirm the gene expression patterns,semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out for 3 of the randomly picked differentially expressed genes. RESULTS:Within these esophageal cancer deregulated genes,4 patterns of expression were observed at 3 stages embryonic d 11.5 (E11.5),embryonic d 13.5 (E13.5) and postnatal d1 (P1).(1) Up-regulation during the E11.5 period, down-regulation during the E13.5 and P1 period (up-down- down),the 10 up-regulated genes during the E11.5 period could be classified into 6 known genes and 4 unknown genes. The known genes included differentiation related genes (S100A8),immunity related gene (IGL),translation and transcription regulation genes (RPL15,EEF1A1),cytoskeletal protein (TUBA1),cysteine protease inhibitor (cystatin B). (2) Up-regulation during the E13.5 and P1 period (down- up-up),such as the SPRR2A which was down-regulated at E11.5.(3) Down-regulation during the E11.5 and E13.5 period (down-down-up),such as RHCG and keratin 4.(4) Fluctuating expression,down initially,up at E13.5,and then down again (down-up-down).EMP1 belonged to such a gene,which was highly expressed at E13.5. CONCLUSION:The results will be helpful for understanding the function of esophageal squamous cell carcinoma (ESCC) deregulated genes in embryonic development and tumorigenesis.S100A8 and S100A9 may play different roles in early embryonic development.IGL may be an oncofetal protein,and EMP1 relates with neurogenesis at E13.5.The genes identified pertinent to embryonic development may serve as candidate susceptibility genes for inherited esophageal cancer disorders as well as for various heritable disorders of embryonic development.

Cytotoxic effect of a non-peptidic small molecular inhibitor of the p53-HDM2 interaction on tumor cells

AIM: To investigate if non-peptidic small molecular inhibitors of the p53-HDM2 interaction could restore p53 function and kill tumor cells.METHODS: A series of non-peptidic small HDM2 inhibitors were designed by computer-aided model and synthesized by chemical method. Syl-155 was one of these inhibitors. Cytotoxic effect of syl-155 on three tumor cell lines with various states of p53, HT1080 (wild-type p53), KYSE510 (mutant p53), MG63 (p53 deficiency) was evaluated by MTT assay, Western blot and flow cytometry.RESULTS: Syl-155 stimulated the accumulation of p53 and p21 protein in HT1080 cells expressing wild-type p53, but not in KYSE510 and MG63 cells. Consequently, syl-155 induced cell cycle arrest and apoptosis in HT1080 cells.CONCLUSION: Non-peptidic small molecular inhibitors of the p53-HDM2 interaction show promise in treatment of tumors expressing wild-type p53.

Overexpression of ETS2 in human esophageal squamous cell carcinoma

AIM: To study the expression pattern of ETS2 (erythroblastosisvirus oncogene homolog 2) in human esophageal squamouscell carcinoma (ESCC).METHODS: Reverse transcription polymerase chain reaction(RT-PCR) and Northern blot were performed to examinethe expression level of ETS2 mRNA in 37 pairs of ESCCtissue samples. Western blot and immunohistochemistrywere carried out to check the expression level of ETS2 proteinin 30 pairs of ESCC tissue specimens.RESULTS: RT-PCR and Northern blot analysis showed thatETS2 mRNA upregulated in 75.7 % (28/37) examined ESCCtissues relative to matched normal tissues. From those 37cases, 14 cases were randomly selected to perform Westernblot and the results revealed that ETS2 protein overexpressedin 71.4 % (10/14) checked ESCC tissues compared with thecorresponding normal tissues. Moreover, the expressionpatterns of ETS2 protein in those 14 cases were identical tothose of ETS2 mRNA displayed by RT-PCR or Northern Blot.Immunohistochemistry analysis showed that the expressionlevel of ETS2 protein rose in 75 % (12/16) tumor epithelialcells contrasted to the normal cells. Altogether the expressionlevel of ETS2 protein increased in 73.3 % (22/30) checkedESCC tissue samples contrary to their normal counterparts.CONCLUSION: The results suggested that ETS2overexpressed in paired human ESCC tissue samples at bothmRNA and protein levels and may be associated with thetumorigenesis of esophagus.

Adenovirus-mediated Transfer of p53 and p16Inhibiting Proliferating Activity of Human Bladder Cancer Cell EJin vitro and in vivo

To evaluate the effects of adenovirus (Ad) - mediated transfer of p5 3and p16 on hum an bladder cancer cells EJ,EJwere transfected with Ad- p5 3and Ad- p16 . Cell growth,m orphologi- cal change,cell cycle,apoptosis were measured using MTT assay,flow cytom etry,cloning form a- tion,im munocytochemical assays.Ad- p16 or Ad- p5 3alone could inhibit the proliferating activity of EJcells in vitro.Ad- p5 3could induce apoptosis of partial EJcells.G1arrest was observed72 h after infection with Ad- p16 ,but apoptosis was not obvious.The transfer of Ad- p16 and Ad- p5 3 could significantly inhibit the growth of EJcells,decrease the cloning formation rate and induce apoptosis of large num ber of EJcells. The occurrence time of subcutaneous tumor was delayed and the tum or volume in 4 weeks was dim inished by using Ad- p5 3com bined with Ad- p16 and the dif- ference was significant com pared with using Ad- p5 3or Ad- p16 alone.It was suggested that the transfer of wild- type p5 3and p16 could significantly inhibit the growth of human bladder cancer in vitro and in vivo.