Transcriptome Profiling of Peripheral Blood Cells Identifies Potential Biomarkers for Doxorubicin Cardiotoxicity in a Rat Model

<div><h3>Aims</h3><p>Doxorubicin (DOX), a widely used anticancer agent, can cause an unpredictable cardiac toxicity which remains a major limitation in cancer chemotherapy. There is a need for noninvasive, sensitive and specific biomarkers which will allow identifying patients at risk for DOX-induced cardiotoxicity to prevent permanent cardiac damage. The aim of this study was to investigate whether the expression of specific genes in the peripheral blood can be used as surrogate marker(s) for DOX-induced cardiotoxicity.</p> <h3>Methods/Results</h3><p>Rats were treated with a single dose of DOX similar to one single dose that is often administered in humans. The cardiac and peripheral blood mononuclear cells (PBMCs) genome-wide expression profiling were examined using Illumina microarrays. The results showed 4,409 differentially regulated genes (DRG) in the hearts and 4,120 DRG in PBMC. Of these 2411 genes were similarly DRG (SDRG) in both the heart and PBMC. Pathway analysis of the three datasets of DRG using Gene Ontology (GO) enrichment analysis and Ingenuity Pathways Analysis (IPA) showed that most of the genes in these datasets fell into pathways related to oxidative stress response and protein ubiquination. IPA search for potential eligible biomarkers for cardiovascular disease within the SDRG list revealed 188 molecules.</p> <h3>Conclusions</h3><p>We report the first in-depth comparison of DOX-induced global gene expression profiles of hearts and PBMCs. The high similarity between the gene expression profiles of the heart and PBMC induced by DOX indicates that the PBMC transcriptome may serve as a surrogate marker of DOX-induced cardiotoxicity. Future directions of this research will include analysis of PBMC expression profiles of cancer patients treated with DOX-based chemotherapy to identify the cardiotoxicity risk, predict DOX-treatment response and ultimately to allow individualized anti-cancer therapy.</p> </div

Estimated log2 fold change for the 2,411 SDRG (26.4% of those expressed) that were identified as significant in both heart and PBMC tissues.

<p>DOX treatment down-regulated most gene expression with down-regulation stronger in PBMC.</p

Graphical representation of NRF2-mediated oxidative stress response pathway (A), protein ubiquitination pathway (B) and PI3K/AKT canonical pathways (C), (IPA), overlaid for cardiovascular disease.

<p>Green-notes indicate down-regulated gene.</p

Lymphocytopenia (A) and thrombocytopenia (B) in rats 48 hours after administration of 12 mg/kg DOX.

<p><i>P</i><0.05.</p

Most significant networks of SDRG associated with cardiac damage generated on the basis of the evidence stored in the IPA library.

<p>The top scoring networks identified as “Cardiovascular system development and function; cellular growth and proliferation; post-translational modification” (score 43, focus molecules 35), and “Cell death; cardiovascular system development and function; embryonic development” (score 38, focus molecule 33) were merged and overlaid for cardiovascular disorders.</p

Global functional analysis and comparison of the three datasets.

<p>The significance value associated with a function in Global Analysis is a measure of probability that genes from the dataset under investigation participate in that function. (A) Bio-function analysis and comparison; (B) Tox-function analysis and comparison.</p

P-value plot for the null hypothesis of no difference in gene expression between heart tissues from DOX treated rats versus heart tissues from control rats (A) and PBMC tissues from DOX treated rats versus PBMC tissues from control rats (B).

<p>P-value plot for the null hypothesis of no difference in gene expression between heart tissues from DOX treated rats versus heart tissues from control rats (A) and PBMC tissues from DOX treated rats versus PBMC tissues from control rats (B).</p

Quantitative real-time PCR validation of 9 genes that were differentially regulated in hearts (A) and PBMC (B) of rats 48 hours after treatment with DOX.

<p>Quantitative real-time PCR validation of 9 genes that were differentially regulated in hearts (A) and PBMC (B) of rats 48 hours after treatment with DOX.</p

DOX concentration in hearts (A) and blood (B) of rats 48 hours following administration of 12 mg/kg DOX.

<p><i>P<0.01</i>.</p

Global functional analysis of the dataset of genes differentially regulated by DOX treatment filtered for cardiovascular diseases.

<p>The significance is expressed as a p-value which was calculated using the right-tailed Fisher's Exact Test. (A) Bio-function analysis; (B) Tox-function analysis.</p