Whole cell profiling and identification of galectin-1 as a potential marker of renal cell carcinoma

For most cancers, the patient's prognosis improves dramatically if the disease is detected at an early stage. Although advancements in imaging technology have improved early detection, many cancers remain undetected until it is too late for curative intervention. We have established, for the first time, expression difference mapping analysis of whole cell proteins from renal cell carcinoma (RCC) cell lines using ProteinChip technology. A total of 20 different RCC cell lines were cultured in vitro directly on ProteinChip arrays for 24 h. Direct MS analysis of proteins from the attached cells showed identical protein profiles by all analysed RCC lines. Comparative on‐chip analysis of isolated malignant cells from native tumour specimens revealed protein patterns highly similar to those from the continuous RCC lines. However, cultured primary cortex cells showed specific protein differences. Differential protein profiling of isolated cytosolic and enriched membrane fractions from the RCC lines revealed that the protein pattern of the membrane proteins included or were identical to those of the entire cells. Proteomics analysis of the chip‐binding membrane fractions allowed the identification of three forms of galectin‐1 as potential RCC marker. ProteinChip analysis with a bound‐specific antibody certified that galectin‐1 could be an RCC marker. Immunostaining methods confirmed the overexpression of galectin‐1 in renal carcinoma in comparison to healthy tissue.     

Quantitative proteomic analysis of a paired human liver healthy versus carcinoma cell lines with the same genetic background to identify potential hepatocellular carcinoma markers

To comprehensively measure global changes in protein expression associated with human hepatocellular carcinoma (HCC), comparative proteomic analysis of two cell lines derived from the healthy and carcinoma tissue of a same donor respectively was conducted using quantitative amino acid-coded mass tagging /stable isotope labeling with amino acids in cell culture-based LC-MS/MS approach. Among a total of 501 proteins precisely quantified, the expressions of 128 proteins were significantly altered including 70 proteins up-regulated and 58 down-regulated in HCC cells. According to their previously characterized functions, the differentially expressed proteins were found associated with nine functional categories including glycolysis, stress response, cell communication, cell cycle, apoptosis/death, etc. For example, multiple enzymes involving glycolysis pathway were found differentially regulated in HCC cells, illustrating the critical participation of glycolysis in the HCC transformation. The accuracy of certain differentially expressed proteins identified through the amino acid-coded mass tagging-based quantification was validated in the paired cell lines, and later their pathological correlations were examined in multiple clinical pairs of normal versus tumor tissues from HCC specimen by using a variety of biological approaches including Western blotting and in situ immunoassays. These consistencies suggested that multiple proteins such as HSP27, annexin V, glyceraldehyde-3-phosphate dehydrogenase, nucleolin and elongation factor Tu could be the biomarkers candidates for diagnosis of HCC.     

Discovery of stage-related proteins in esophageal squamous cell carcinoma using proteomic analysis

Esophageal squamous cell carcinoma (ESCC) is the major subtype of esophageal cancers in China, and characterized with high morbidity and mortality. So far, the diagnosis of ESCC is mainly dependent on the alterations in esophageal histology, but most cases of ESCC with low stage do not display visible histological abnormalities. Therefore, a deep understanding of the mechanism of ESCC progression and seeking stage-specific molecules might improve the diagnosis and therapy for ESCC. In this study, we used proteomics to analyze ESCC tissues with classification by TNM stage, and determined the proteomic features correlated with ESCC progression (from stages I to III). Proteins that exhibited significantly different expression patterns between ESCC and corresponding normal esophageal tissues were identified using MS. The identified proteins with differentiated expression mainly fell into three protein categories (i.e. cytoskeleton system-associated proteins, metabolism enzymes, and heat shock proteins). In addition, real-time PCR highlighted some molecules that were associated with tumor stages at the mRNA level, such as enolase 1, chromosome 1 ORF 10, elastase inhibitor, α B crystalline, stress-induced phosphoprotein 1, and squamous cell carcinoma antigen 1. Altogether, these data provided further information on ESCC progression and potential drug targets for ESCC clinical therapy.     

Expression of tropomyosin alpha 4 chain is increased in esophageal squamous cell carcinoma as evidenced by proteomic profiling by two-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry

To identify proteins associated with esophageal carcinogenesis, we performed protein profiling of 16 esophageal squamous cell carcinomas (ESCCs) and paired noncancerous tissues by 2-DE and MS/MS. In cancerous tissues, three spots showed significant up-regulation in the amount of protein, while eight spots were significantly down-regulated. The identities of the spots were determined by PMF with LC-MS/MS and were confirmed by immunoblotting. The up-regulated proteins were tropomyosin alpha 4 chain, transgelin, and pyruvate kinase. The down-regulated proteins were serum albumin precursor, isoforms of annexin A1, tropomyosin beta chain, 14-3-3 protein sigma, and isoforms of serotransferrin precursor. In all 16 cases, up-regulation of the tropomyosin alpha 4 chain was confirmed by immunoblotting. Localization of the tropomyosin alpha 4 chain in ESCC cells and adjacent fibroblasts was confirmed by immunohistochemistry.     

Comparative proteomic analysis of differentially expressed proteins in an in vitro cellular carcinogenesis model of oral squamous cell carcinoma

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2‐DE and LC‐tandem mass chromatography to separate and identify differentially expressed proteins. Forty‐five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.     

Isoform-specific expression and characterization of 14-3-3 proteins in human glioma tissues discovered by stable isotope labeling with amino acids in cell culture-based proteomic analysis

Human 14-3-3 proteins have isoform-specific expression and functions in different kinds of normal or tumor cells and tissues. However, the expression profiling of 14-3-3 proteins and isoform-specific biological functions are unclear in human glioma so far. In our study, the expression levels and characterization of 14-3-3 isoforms in human glioma tissues were investigated by a sensitive, accurate stable isotope labeling with amino acids in cell culture-based quantitative proteomic strategy. As a result, except unexpressed 14-3-3σ, the other six isoforms, with different expression levels, were existed in glioma tissues and para-cancerous brain tissues (PBTs). 14-3-3β and η were upregulated, whereas 14-3-3ζ was downregulated in glioma tissues compared with that in PBTs. And the other three isoforms 14-3-3ε, θ, and γ had similar expression levels in human glioma tissues and PBTs. Western blot and immunohistochemistry analysis were both consistent with the quantitative proteomic data. The loss of expression of 14-3-3σ was further discovered due to DNA high methylation in its coding region in glioma by methylation-specific PCR analysis. These results indicated that the four isoforms, including 14-3-3β, η, ζ, and σ, may play important roles in tumorigenesis of human glioma, which is probably used as potential biomarkers for diagnosis and targets for treatment of human gliomas in future.