Analysis of candidate genes through a proteomics-based approach in primary cell lines from malignant melanomas and their metastases

Abstract

Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.

Silencing of Peroxiredoxin 2 and Aberrant Methylation of 33 CpG Islands in Putative Promoter Regions in Human Malignant Melanomas

“…Although Prx II is a 2-Cys thioredoxin peroxidase and a cellular antioxidant, it interacts with activated PDGF receptor h, unlike other peroxiredoxins, and suppresses its phosphorylation (19). Down-regulation of Prx II was recently shown to be associated with progression of melanomas (20) and urinary bladder cancers (21). Molecular mechanisms for the down-regulation have been unknown, but our study here showed that gene silencing by promoter methylation is one of the mechanisms.…”

Peroxiredoxin-2 Represses Melanoma Metastasis by Increasing E-Cadherin/β-Catenin Complexes in Adherens Junctions

“…Among these enzymes, Prx is the most abundant enzyme and is known to be involved in receptor tyrosine kinase-mediated signal transduction (12). Interestingly, it has recently been shown that one of the Prx family members Prx2 (gene loci, PRDX2), which belongs to 2-cys Prx subfamily and reduces the peroxides in the presence of NADPH by coupling with thioredoxin/thioredoxin reductase system, is downregulated in melanoma cell lines and particularly in metastatic malignant types (13,14). However, the function of the Prx2 enzyme in the melanoma cell growth and metastasis remains unknown.…”

Primer on the human genome

“…Many of the proteins that were found to be more abundant in tumor samples were stress response proteins, including multiple heat shock proteins. 111 A similar study demonstrated high levels of hepatocytederived growth factor levels in tumor samples. 112 In an effort to examine the mechanism of the antitumor effect of interferon alfa, the proteomes of two melanoma cell lines, one interferon-sensitive, the other interferon-resistant, were compared by use of 2D electrophoresis and mass spectroscopy.…”