Proteomic analysis identifies MMP-9, DJ-1 and A1BG as overexpressed proteins in pancreatic juice from pancreatic ductal adenocarcinoma patients

Abstract

Background: There is an urgent need to discover more sensitive and specific biomarkers to improve early diagnosis and screen high-risk patients for pancreatic ductal adenocarcinoma (PDAC). Pancreatic juice is an ideal specimen for PDAC biomarkers discovery, because it is an exceptionally rich source of proteins released from pancreatic cancer cells.

Methods: To identify novel potential biomarkers for PDAC from pancreatic juice, we carried out difference gel electrophoresis (DIGE) and tandem mass spectrometry (MS/MS) to compare the pancreatic juice profiling from 9 PDAC patients and 9 cancer-free controls. Of the identified differently expressed proteins, three up-regulated proteins in pancreatic cancer juice, matrix metalloproteinase-9 (MMP-9), oncogene DJ1 (DJ-1) and alpha-1B-glycoprotein precursor (A1BG), were selected for validation by Western blot and immunohistochemistry. Serum MMP-9 levels were also detected by enzyme linked immunosorbent assay (ELISA).

Results: Fourteen proteins were up-regulated and ten proteins were down-regulated in cancerous pancreatic juice compared with cancer-free controls. Increased MMP-9, DJ-1 and A1BG expression in cancerous pancreatic juice were confirmed by Western blot. Immunohistochemical study showed MMP-9, DJ-1 and A1BG positively expressed in 82.4%, 72.5% and 86.3% of pancreatic cancer tissues, significantly higher than that in normal pancreas tissues. Up-regulation of DJ-1 was associated with better differentiation (p < 0.05). Serum MMP-9 levels were significantly higher in PDAC (255.14 ng/ml) than those in chronic pancreatitis (210.22 ng/ml, p = 0.009) and healthy control (203.77 ng/ml, p = 0.027).

Conclusion: The present proteome analysis revealed MMP-9, DJ-1 and A1BG proteins as elevated in pancreatic juice from PDAC, which suggest their further utility in PDAC diagnosis and screening. This is the first time A1BG was identified as a potential biomarker in pancreatic cancer associated samples. The measurement of serum MMP-9 might be clinically useful for PDAC diagnosis.

Figure 1

Representative gel images of proteins extracted from pancreatic cancer juice and cancer-free controls juice. Representative 2-DE gel images of pancreatic juice proteins from PDAC (A) and cancer-free controls (B), and representative DIGE overlay image (C). Labeled spots are significantly up-regulated proteins in pancreatic cancer juice (A), in cancer-free controls juice (B) and a total of 24 differentially expressed protein spots (C).

Figure 2

Individual differentially expressed protein spots in pancreatic cancer juice and cancer-free controls juice. Selected areas of 14 up-regulated (A) and 10 down-regulated (B) spots and their corresponding DIGE images.

Figure 3

MMP-9, DJ-1 and A1BG expression analysis by Western blot. Increased MMP-9 (A), DJ-1 (B) and A1BG (C) were detected in the cancerous juice samples compared with cancer-free pancreatic juice samples; MMP-9 expression evinced both 92 kDa and 82 kDa bands, corresponding to the latent and activated forms of MMP-9, respectively.

Figure 4

MMP-9, DJ-1 and A1BG expression analysis by Immunohistochemistry (×200). MMP-9 (A), DJ-1 (C) and A1BG (E) over-expressed in PDAC tissues. MMP-9 (B), DJ-1(D) and A1BG (F) were not detectable in normal pancreas tissues.

Figure 5

Box plot of MMP-9 serum levels for PDAC, chronic pancreatitis and normal controls by ELISA. The serum levels of MMP-9 in PDAC patients were significantly higher than those in chronic pancreatitis patients and in healthy controls (p < 0.05). PDAC: pancreatic ductal adenocarcinoma patients; CP: chronic pancreatitis patients; N: healthy controls.