Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Mar 23;6(3):e17177.
doi: 10.1371/journal.pone.0017177.

Metabolites of purine nucleoside phosphorylase (NP) in serum have the potential to delineate pancreatic adenocarcinoma

Affiliations

Metabolites of purine nucleoside phosphorylase (NP) in serum have the potential to delineate pancreatic adenocarcinoma

Shaiju K Vareed et al. PLoS One. .

Abstract

Pancreatic Adenocarcinoma (PDAC), the fourth highest cause of cancer related deaths in the United States, has the most aggressive presentation resulting in a very short median survival time for the affected patients. Early detection of PDAC is confounded by lack of specific markers that has motivated the use of high throughput molecular approaches to delineate potential biomarkers. To pursue identification of a distinct marker, this study profiled the secretory proteome in 16 PDAC, 2 carcinoma in situ (CIS) and 7 benign patients using label-free mass spectrometry coupled to 1D-SDS-PAGE and Strong Cation-Exchange Chromatography (SCX). A total of 431 proteins were detected of which 56 were found to be significantly elevated in PDAC. Included in this differential set were Parkinson disease autosomal recessive, early onset 7 (PARK 7) and Alpha Synuclein (aSyn), both of which are known to be pathognomonic to Parkinson's disease as well as metabolic enzymes like Purine Nucleoside Phosphorylase (NP) which has been exploited as therapeutic target in cancers. Tissue Microarray analysis confirmed higher expression of aSyn and NP in ductal epithelia of pancreatic tumors compared to benign ducts. Furthermore, extent of both aSyn and NP staining positively correlated with tumor stage and perineural invasion while their intensity of staining correlated with the existence of metastatic lesions in the PDAC tissues. From the biomarker perspective, NP protein levels were higher in PDAC sera and furthermore serum levels of its downstream metabolites guanosine and adenosine were able to distinguish PDAC from benign in an unsupervised hierarchical classification model. Overall, this study for the first time describes elevated levels of aSyn in PDAC as well as highlights the potential of evaluating NP protein expression and levels of its downstream metabolites to develop a multiplex panel for non-invasive detection of PDAC.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Workflow for Proteomic Profiling of Pancreatic Juice Proteome.
Pancreatic juice specimens from 25 patients (7 benign and 18 PDAC) were collected in tubes containing protease inhibitors. The proteome was subjected to pre-fractionation using either SDS-PAGE or SCX-fractionation. For 1D-SDS-PAGE based fractionation, 100 µg of pancreatic juice proteome was electrophoresed on a 8–16% SDS-PAGE and the gel was excised into 30 equal pieces and subjected to in-gel trypsin digestion as described in the methods section. For SCX fractionation, 200 µg of pancreatic juice proteome was first subjected to in-solution trypsin digestion followed by separation of peptides into 20 fractions. Peptides derived from both these methods were separated using reverse phase chromatography on a 43 mm HPLC-Chip and analyzed using an Agilent XCT-ultra ion trap or 6510 QTOF mass spectrometer. The raw spectral files were converted into mzXML format and searched with X!Tandem using a Human IPI-database (see methods for details). The identified peptides and proteins were curated using PeptideProphet and ProteinProphet and the class-specific protein signatures were obtained using coupled to t-test and FDR correction (see methods for details). A subset of PDAC-associated proteins and NP were examined by tissue microarrays and immunblot analysis to confirm their PDAC-specific overexpression in tissues, while expression of NP and its downstream metabolites were examined in serum specimens using immunoblot analysis and targeted LC-MS respectively.
Figure 2
Figure 2. Over view of pancreatic juice proteome and validation of PARK-7 and SNCA expression in PDAC and benign tissues.
A, Heat map representation of the 56 significantly altered proteins (P<0.05 & FDR<12%) in 25 pancreatic juice specimens (7 benign, 2 CIS and 16 PDAC). Columns represent samples and rows refer to proteins. Shades of yellow represent elevated expression of the protein and shades of blue indicate decrease in the protein levels relative to its median expression value (see color scale). B, immunoblot confirmation of elevated PARK-7 (DJ-1) expression in PDAC tissues. C, representative photomicrograph of the TMA showing immunostaining of aSyn in benign pancreas D, same as C but in PDAC E, Higher magnification of D showing aggregated staining pattern for aSyn in PDAC F, quantification of TMA staining for aSyn in benign (n = 11) and PDAC samples (n = 67). Magnification of A and B: 40X, for C: 100X.
Figure 3
Figure 3. Validation of elevated expression of Nucleoside Phosphorylase (NP) and levels of its regulated metabolites in PDAC.
A, Immunoblot confirmation of elevated NP expression in PDAC tissues. B, representative photomicrograph of the TMA showing immunostaining of NP in benign pancreas C, same as B but in PDAC D, quantification of TMA staining for NP in benign (n = 11) and PDAC samples (n = 49). Magnification of B and C: 40X E, immunoblot showing predominant expression of a doublet at ∼32 KDa immunoreactive to NP antibody in PDAC sera. +control: 10 µg total protein from PANC-1 F, immunoblot-based quantification of NP levels in benign and PDAC sera G, Unsupervised hierarchical classification of PDAC (n = 24) and benign (n = 24) specimens based on the relative levels of 7 NP regulated metabolites in serum. Columns, samples; rows, metabolites. Asterisks denote metabolites individually correlated (P<0.05, t-test) with PDAC/benign status.

Similar articles

Cited by

References

    1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. Cancer statistics, 2009. CA Cancer J Clin. 2009;59:225–249. - PubMed
    1. Warshaw AL, Fernandez-del Castillo C. Pancreatic carcinoma. N Engl J Med. 1992;326:455–465. - PubMed
    1. Goggins M, Canto M, Hruban R. Can we screen high-risk individuals to detect early pancreatic carcinoma? J Surg Oncol. 2000;74:243–248. - PubMed
    1. Rosty C, Goggins M. Early detection of pancreatic carcinoma. Hematol Oncol Clin North Am. 2002;16:37–52. - PubMed
    1. Farrell JJ, van Rijnsoever M, Elsaleh H. Early detection markers in Pancreas Cancer. Cancer Biomark. 2005;1:157–175. - PubMed

Publication types

MeSH terms