Cathepsin B Dependent Cleavage Product of Serum Amyloid A1 Identifies Patients with Chemotherapy-Related Cardiotoxicity

Abstract

Improvements in long-term cancer survival rates have resulted in an increase in the prevalence of chemotherapy-linked cardiac failure, but treatment-induced cardiac injuries may not be detected until long after therapy. Monitoring cardiac function is recommended; however, cardiovascular injury in cancer patients differs from those with primary cardiac dysfunction, which limits the utility of traditional cardiac biomarkers. Here we examined plasma levels of peptides produced by cathepsin B, which is released during chemotherapy-induced cardiac injury. We applied nanotrap fractionation to enrich plasma peptides from cancer patients treated with or without chemotherapy. Peptides associated with chemotherapy-induced cardiotoxicity, but not other cardiac injury, were identified by mass spectrometry, and their dependence on cathepsin B activity was determined using enzyme inhibition experiments. We found that a peptide (SAA-1525) derived from serum amyloid A1 was significantly increased in cardiotoxicity patients, and its production was inhibited when plasma samples were pretreated with cathepsin B specific inhibitors. Plasma SAA-1525 also correlated with other markers of cardiac injury. Analysis of plasma SAA-1525 levels may hold potential as a rapid and minimally invasive method to monitor subclinical injury, thereby allowing timely intervention to mitigate further cardiac damage and avoid more severe clinical presentation.

Figure 1

Rational progression of the discovery of cardiotoxicity biomarkers from sample preparation to identification of cardiotoxicity specific peptide in human biological fluids.

Figure 2

Peptide SAA-1525 identification. (A) SAA-1525 LC-MS signal normalized to spiked-in ACTH fragment (Mean ± SE; **, p < 0.01 by two-way ANOVA). (B) ROC analysis for SAA-1525 differentiation of Ctox and nCtox patients. (C) SAA-1525 peptide peak LC-MS/MS sequence. (D) CTSB cleavage sites in SAA1. Blue arrows indicate CTSB cleavage sites. * indicates a cleavage site not assigned to CTSB activity.

Figure 3

SAA-1525 production is CSTB-dependent. ACTH-normalized LC-MS signal for cleavage of recombinant SAA1 to (A) SAA-1525 and (B) SAA-2126 and (C) cleavage of synthetic SAA-2126 peptide to SAA-1525 in human plasma with high and low CTSB levels. Mean ± SE; ****, p < 0.0001 by Student’s t test. (D) Cleavage of SAA1 to SAA-1525 in human plasma treated with or without the CTSB-specific inhibitors E64 and ALLM.

Figure 4

Radar chart representing the correlation between laboratory indicators and (A) SAA-1525 or (B) CTSB. Each axis displays the laboratory indicators and the values are Spearman r.