Platelet-protein-profiling of healthy controls and patients with early- and late-stage colorectal cancers

Abstract

Introduction: Colorectal cancer (CRC) is one of the most frequent malignancies in the western world. Early tumor detection and intervention are important determinants on CRC patient survival. During tumor dissemination, growth and angiogenesis, platelets actively and selectively store and segregate proteins. Hence, the platelet protein content is a potential marker of early tumor growth. In order to screen for innovative, diagnostic and prognostic biomarkers, we identified differentially regulated proteins within platelets distinguishing healthy controls from patients with early- and latestage CRCs. Material(s) and Method(s): We compared protein profiles of platelets between healthy volunteers (n = 12) and patients with early- (n = 7) and late-stage (n = 5) CRCs by using multiplex-fluorescence two-dimensional gel electrophoresis (2-DE). Results with differences in protein levels between these groups were analyzed with SameSpot software followed by Principle Component Analysis (PCA). Proteins of interest were identified by Mass Spectrometry (MS) and subjected to Ingenuity Pathway Analysis (IPA). Target proteins were validated and confirmed by fluorescence-based Western Blot analyses using an independent collective of platelet protein samples (healthy controls, n = 12; early CRC, n = 5; late CRC, n = 6). Result(s): By inter-group comparison (p < 0.05), 39 differentially expressed protein spots were detected. A PCA-based cluster analysis showed clustering and good separation between the three groups. 31 proteins were identified by MS and IPA resulted in one high-ranked network maintaining 14 proteins. Of those, Caspase 3 (CASP3), Clusterin (CLU) and Vinculin (VCL) were down-regulated, whereas Cofilin 1 (CFL1) and Glutathione Synthetase (GSS) were present at higher levels in platelets from CRC patients compared to control individuals. Expression characteristics of these five proteins were confirmed by Western Blot analyses in an independent cohort. Conclusion(s): This study showed differently regulated proteins of platelets between healthy controls and patients with early- and late-stage CRCs. The potential of CASP3, CLU, VCL, CFL1 and GSS as biomarkers for CRC diagnosis and prognosis could improve individualized medicine.