Transcriptome analysis of different multidrug-resistant gastric carcinoma cells.

Multidrug resistance (MDR) of human cancers is the major cause of failure of chemotherapy. To better understand the molecular events associated with the development of different types of MDR, two different multidrug-resistant gastric carcinoma cell lines, the MDR1/P-glycoprotein-expressing cell line EPG85-257RDB and the MDR1/P-glycoprotein-negative cell variant EPG85-257RNOV, as well as the corresponding drug-sensitive parental cell line EPG85-257P, were used for analyses of the mRNA expression profiles by cDNA array hybridization. Of more than 12,000 genes spotted on the arrays, 156 genes were detected as being significantly regulated in the cell line EPG85-257RDB in comparison to the non-resistant cell variant, and 61 genes were found to be differentially expressed in the cell line EPG85-257RNOV Seventeen genes showed a differential expression level in both multidrug-resistant gastric carcinoma variants. The impact of these alterations in gene expression levels in different multidrug-resistant gastric carcinoma cell variants is discussed.

Overexpression of cathepsin B in gastric cancer identified by proteome analysis.

We aimed to validate an analytical approach based on proteomics on gastric cancer specimens for the identification of new putative diagnostic or prognostic markers. Primary screening was performed on gastrectomy specimens obtained from ten consecutive patients with gastric cancer. Gastric epithelial cells were obtained with an epithelial cell enrichment technique, homogenized and then separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The differential protein expression pattern was verified stepwise by Western blotting and immunohistochemistry on samples from 28 and 46 cancer patients, respectively. The putative clinical applicability and prognostic use were tested by an enzyme-linked immunoabsorbent assay on serum samples obtained from 149 cancer patients. One hundred-ninety-one differentially expressed protein spots were found by 2-D PAGE and identified by mass spectrometry, including cathepsin B, which was over-expressed in six (60%) patients. Western blotting confirmed that the active form of cathepsin B is over-expressed, while immunohistochemistry showed strong cytoplasmic staining in cancer tissues of 45 (98%) patients. The serum level of cathepsin B was increased in patients with gastric cancer compared to healthy controls (P = 0.0026) and correlated with T-category and the presence of distant metastases (P < 0.05). Serum levels above 129 pmol x L(-1) were associated with a reduced survival rate (P = 0.0297). Proteome analysis is a valuable tool for the identification of prognostic markers in gastric cancer: Increased cathepsin B serum levels are associated with advanced tumor stages and progressive disease, which enables the classification of some gastric cancer patients into a subgroup that should undergo aggressive therapy.

Epithelial cell preparation for proteomic and transcriptomic analysis in human pancreatic tissue.

Standardized sample preparation procedures constitute a prerequisite for obtaining reliable and reproducible results in gene expression research in humans. In particular, in diseases such as pancreatic cancer and pancreatitis, isolating epithelial cells is an important step preceding such research. In pancreatic tissue, the high amount of RNAases is a further problem when it comes to obtaining high-quality RNA, and the presence of secreted proteases accelerates protein degradation. We developed a successful method that addresses these different problems. This method, which uses epithelial cell surface antibody Ber-Ep4, proteases, and RNAases inhibitors, leads to a significant enrichment (> 95% purity) of epithelial cells from fresh human tissue samples and allows for both proteomics (Western Blot, 2D PAGE) and transcriptomics studies (rtPCR, cDNA microarray). Compared with other cell purification procedures, this method is characterized by several advantages: a large quantity of cells available for downstream analysis, combined transcriptomics and proteomics studies using the same samples, better reproducibility of proteomics studies, and an acceptable yield (63%) for gene expression arrays studies. Moreover, a quality control protocol addressing the needs of the industry and the requirements of regulatory agencies is proposed.