Exploratory plasma proteomic analysis in a randomized crossover trial of aspirin among healthy men and women

<div><p>Long-term use of aspirin is associated with lower risk of colorectal cancer and other cancers; however, the mechanism of chemopreventive effect of aspirin is not fully understood. Animal studies suggest that COX-2, NFκB signaling and Wnt/β-catenin pathways may play a role, but no clinical trials have systematically evaluated the biological response to aspirin in healthy humans. Using a high-density antibody array, we assessed the difference in plasma protein levels after 60 days of regular dose aspirin (325 mg/day) compared to placebo in a randomized double-blinded crossover trial of 44 healthy non-smoking men and women, aged 21–45 years. The plasma proteome was analyzed on an antibody microarray with ~3,300 full-length antibodies, printed in triplicate. Moderated paired t-tests were performed on individual antibodies, and gene-set analyses were performed based on KEGG and GO pathways. Among the 3,000 antibodies analyzed, statistically significant differences in plasma protein levels were observed for nine antibodies after adjusting for false discoveries (FDR adjusted p-value<0.1). The most significant protein was succinate dehydrogenase subunit C (SDHC), a key enzyme complex of the mitochondrial tricarboxylic acid (TCA) cycle. The other statistically significant proteins (NR2F1, MSI1, MYH1, FOXO1, KHDRBS3, NFKBIE, LYZ and IKZF1) are involved in multiple pathways, including DNA base-pair repair, inflammation and oncogenic pathways. None of the 258 KEGG and 1,139 GO pathways was found to be statistically significant after FDR adjustment. This study suggests several chemopreventive mechanisms of aspirin in humans, which have previously been reported to play a role in anti- or pro-carcinogenesis in cell systems; however, larger, confirmatory studies are needed.</p></div

Sequences of primers and probes used for QPCR.

<p>* The Artus EBV TM PCR kit primer and probes are proprietary and not made publicly available.</p><p>Sequences of primers and probes used for QPCR.</p

Sequences of primers and probes used for QPCR.

<p>* The Artus EBV TM PCR kit primer and probes are proprietary and not made publicly available.</p><p>Sequences of primers and probes used for QPCR.</p

Advantages and limitations of molecular analyses.

<p>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118989#pone.0118989.ref046" target="_blank">46</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118989#pone.0118989.ref063" target="_blank">63</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118989#pone.0118989.ref064" target="_blank">64</a>].</p><p>Advantages and limitations of molecular analyses.</p

Demographic characteristics of 44 participants.

<p>Demographic characteristics of 44 participants.</p

Patient and tumour characteristics.

<p>Patient and tumour characteristics.</p

Meta-analysis of CMV positivity in breast cancer tissue samples.

<p>Random effects meta-analysis was performed on the proportions of samples that were positive for CMV. Proportions had 95% confidence intervals derived from the Normal approximation to the binomial with 0.5 added to zero counts. Estimates of the average proportion positive were calculated for all studies and for each assay type that was used in multiple studies. Results of studies that analyzed breast tissue samples for CMV are shown according to the method of analysis. The overall strength of association for each type of analysis, and the overall strength of association for all studies are shown at the bottom of the figure. There was considerable heterogeneity in the CMV analyses (I² = 99.6%). Footnote to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118989#pone.0118989.g002" target="_blank">Fig. 2</a>: PCR results for Harkins et al were not included in this meta-analysis because nested PCR was performed on only 8 specimens, all of which were positive on IHC (please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118989#pone.0118989.s001" target="_blank">S1 Table</a>).</p

Results of QPCR analysis.

<p>Results of QPCR analysis.</p

Proteins that differed significantly between aspirin and placebo periods with false discovery rate (FDR) <0.10.

<p>Proteins that differed significantly between aspirin and placebo periods with false discovery rate (FDR) <0.10.</p

Meta-analysis of EBV positivity in breast cancer tissue samples.

<p>Random effects meta-analysis was performed on the proportions of samples that were positive for EBV rather than relative risks, because only six studies (including ours) included paired normal samples. Proportions had 95% confidence intervals derived from the Normal approximation to the binomial with 0.5 added to zero counts. Estimates of the average proportion positive were calculated for all studies and for each assay type that was used in multiple studies. Results of studies that analyzed breast tissue samples for EBV are shown according to the method of analysis. The overall strength of association for each type of analysis, and the overall strength of association for all studies are shown at the bottom of the figure. There was considerable heterogeneity in the EBV analyses (I² = 98.6%).</p