Identification of Differentially Expressed Proteins in the Serum of Colorectal Cancer Patients Using 2D-DIGE Proteomics Analysis

Early detection of colorectal cancer (CRC) is vital for the improvement of disease prognosis. However to date there are no blood-based biomarkers sensitive and specific enough for early diagnosis. We analysed the differences in serum protein expression of early stage CRC (Dukes’ A and B) and late stage CRC (Dukes’ C and D) against normal controls using 2D Fluorescence Difference Gel Electrophoresis (2D-DIGE). Analysis of the 2D maps showed that 23 proteins were differentially expressed between groups (p≤0.05) and these proteins were identified with LC-MS/MS. Eight proteins were up-regulated and 2 down-regulated in patients with early CRC, whereas 14 proteins were up-regulated and 4 downregulated in those with late CRC compared to normal controls (p≤0.05). Five proteins, namely apolipoprotein A1 (APOA1), apolipoprotein E (APOE), complement factor H (CFH), galectin-7 (GAL7) and synaptojanin-2 (SYNJ2) were validated using ELISA and only APOA1 and GAL-7 showed consistent findings. Further validation using immunohistochemistry showed negative immunoreactivity for GAL-7 in CRC tissues, suggesting that GAL-7 detected in the serum did not originate from the CRC tumour. APOA1 showed positive immunoreactivity but its expression did not correlate with Dukes’ staging (p=0.314), tumour grading (p=0.880) and lymph node involvement (p=0.108). Differences in APOA1 isoforms and/or conformation between serum and tissue samples as well as tumour heterogeneity may explain for the discrepancies between DIGE and ELISAwhen compared to immunohistochemistry. Structural and functional studies of APOA1 in future would best describe the role of APOA1 in CRC.