Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling

• Published in: Proceedings of the National Academy of Sciences - PNAS 2014-05, Vol.111 (21), p.7594-7599
• Main Authors: Wiita, Arun P., Hsu, Gerald W., Lu, Chuanyi M., Esensten, Jonathan H., Wells, James A.
• Format: Article
• Language: English
• Subjects: Activating Transcription Factor 6 - blood; Antineoplastic Combined Chemotherapy Protocols - pharmacology; Apoptosis; Apoptosis - drug effects; Biological markers; Biological Sciences; Biomarkers, Tumor - blood; Blood; Blood plasma; Cancer; Cell death; Cells; Chemotherapy; Enzyme-Linked Immunosorbent Assay; Enzymes; Hematologic neoplasms; High-Temperature Requirement A Serine Peptidase 2; Humans; Mass Spectrometry; Mitochondrial Proteins - blood; Peptide Fragments - blood; Peptides; Proteins; Proteolysis - drug effects; Proteomics; Serine Endopeptidases - blood; Tumors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1405987111

It is known that many chemotherapeutics induce cellular apoptosis over hours to days. During apoptosis, numerous cellular proteases are activated, most canonically the caspases. We speculated that detection of proteolytic fragments released from apoptotic cells into the peripheral blood may serve as a unique indicator of chemotherapy-induced cell death. Here we used an enzymatic labeling process to positively enrich free peptide α-amines in the plasma of hematologic malignancy patients soon after beginning treatment. This N-terminomic approach largely avoids interference by high-abundance proteins that complicate traditional plasma proteomic analyses. Significantly, by mass spectrometry methods, we found strong biological signatures of apoptosis directly in the postchemotherapy plasma, including numerous caspase-cleaved peptides as well as relevant peptides from apoptotic and cell-stress proteins second mitochondria-derived activator of caspases, HtrA serine peptidase 2, and activating transcription factor 6. We also treated hematologic cancer cell lines with clinically relevant chemotherapeutics and monitored proteolytic fragments released into the media. Remarkably, many of these peptides coincided with those found in patient samples. Overall, we identified 153 proteolytic peptides in postchemotherapy patient plasma as potential indicators of cellular apoptosis. Through targeted quantitative proteomics, we verified that many of these peptides were indeed increased post- vs. prechemotherapy in additional patients. Our findings reveal that numerous proteolytic fragments are released from dying tumor cells. Monitoring posttreatment proteolysis may lead to a novel class of inexpensive, rapid biomarkers of cell death.