Paula Álvarez-Chaver

Proteomics for discovery of candidate colorectal cancer biomarkers

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in Europe and other Western countries, mainly due to the lack of well-validated clinically useful biomarkers with enough sensitivity and specificity to detect this disease at early stages. Although it is well known that the pathogenesis of CRC is a progressive accumulation of mutations in multiple genes, much less is known at the proteome level. Therefore, in the last years many proteomic studies have been conducted to find new candidate protein biomarkers for diagnosis, prognosis and as therapeutic targets for this malignancy, as well as to elucidate the molecular mechanisms of colorectal carcinogenesis. An important advantage of the proteomic approaches is the capacity to look for multiple differentially expressed proteins in a single study. This review provides an overview of the recent reports describing the different proteomic tools used for the discovery of new protein markers for CRC such as two-dimensional electrophoresis methods, quantitative mass spectrometry-based techniques or protein microarrays. Additionally, we will also focus on the diverse biological samples used for CRC biomarker discovery such as tissue, serum and faeces, besides cell lines and murine models, discussing their advantages and disadvantages, and summarize the most frequently identified candidate CRC markers.

Secreted clusterin in colon tumor cell models and its potential as diagnostic marker for colorectal cancer.

We studied the specific changes of the secreted protein clusterin and its cytoplasmic precursor regarding colorectal tumorigenesis, using in vitro differentiation of Caco-2 cells. In tumor-like stage, we observed an overexpression of both precursor and secreted clusterin, corroborated in the cell line SW-480. Noticeably, SW-620 cells (from a tumoral node, thus with metastatic capacity) did not show overexpression of either precursor or secreted clusterin, suggesting a downregulation related to local metastasis. We further investigated clusterin in serum, finding a significant increase in colorectal cancer patients, with 81% sensitivity, 79% specificity, and an area under the ROC curve of 0.85.

Selection of putative colorectal cancer markers by applying PCA on the soluble proteome of tumors: NDK A as a promising candidate.

Aiming to find new tumor markers for colorectal cancer (CRC), we applied proteomic methodologies to compare the soluble sub-proteome of healthy and tumoral colorectal mucosa. Out of 91 differentially expressed proteins, 23 were selected by principal component analysis (PCA) as the major contributors to the overall difference detected. After MS/MS analysis, 16 proteins were identified. From those, we chose 14-3-3-zeta/delta, retinoblastoma-binding protein 4 (RBBP-4), DJ-1, and nucleoside diphosphate kinase A (NDK A) for further studies, on the basis of their levels and known implication in cancer. Specific immunodetection demonstrated only the NDK A levels allowed to differentiate healthy mucosa from tumor tissue in all the patients. Hence, we used the colon cancer cell line Caco-2 to study the relationship between NDK A and colon cell tumorigenesis, finding it over-expressed in undifferentiated (tumor-like) cells regarding the differentiated ones. Noticeably, we also found increased levels of the NDK A in the secretome of tumor-like cells and, as expected, indications of higher levels of NDK A in the serum of CRC patients. In conclusion, the four validated proteins could constitute a panel of tissue markers for CRC, being the NDK A the most interesting candidate for further serum biomarker studies.

Proteomic Profiling for Colorectal Cancer Biomarker Discovery

Nowadays, the ideal biomarker for colorectal cancer (CRC) has not been found. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) are suitable techniques for searching new biomarkers. In this chapter, we describe methodology for biomarker discovery based on a proteomic approach. In addition, special attention is given to the sample preparation, including protein extraction, fractionation, and cleanup, as we consider this a critical step. Comparing the proteomic profile of tumor and mucosa, we identified the nucleoside diphosphate kinase A (NDKA) protein as a candidate biomarker for CRC. Finally, we validated NDKA with an ELISA kit using serum samples from individuals of a screening cohort. Our results suggest that serum NDKA is a potential biomarker for screening of CRC and premalignant advanced adenomas (AA).

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum

Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.