Elevation of serum fortilin levels is specific for apoptosis and signifies cell death in vivo

Patuma Sinthujaroen, Nattaporn Wanachottrakul, Decha Pinkaew, John R Petersen, Amornrat Phongdara, Melinda Sheffield-Moore, Ken Fujise, Patuma Sinthujaroen, Nattaporn Wanachottrakul, Decha Pinkaew, John R Petersen, Amornrat Phongdara, Melinda Sheffield-Moore, Ken Fujise

Abstract

Background: Billions of cells undergo apoptosis each day in the average normal adult. The ability to readily assess the degree of apoptosis in human diseases is hampered by the lack of sensitive and specific serum biomarkers of apoptosis. Fortilin is a novel prosurvival molecule that protects cells against various noxious stimuli. While fortilin is secreted into the extracellular space under certain conditions, the relationship between the serum concentration of fortilin and the presence and extent of apoptosis in vivo remains unknown.

Methods & results: Using a newly developed fortilin ELISA system, we show here that fortilin exists in the normal human and mouse circulation. We further demonstrate that fortilin serum levels are significantly elevated in patients with solid cancer, in response to anti-cancer chemo- or radiation therapy. The elevation of fortilin serum levels is more robust and sensitive than that of such previously-reported serum biomarkers of apoptosis as fragmented cytokeratin-18, cytochrome c, and nucleosomal DNA. In addition, targeted apoptotic liver damage induced by Jo2 anti-Fas (CD95) antibody consistently and significantly increased serum fortilin levels in C57BL/6J mice. Finally, when challenged by anti-human-Fas IgM antibody, Jurkat leukemic T cells apoptosed and released fortilin into the medium before plasma membrane integrity was compromised.

Conclusions: Taken together, these data suggest that serum fortilin levels reflect the degree and extent of apoptosis occurring in vivo.

General significance: Fortilin is a viable serum biomarker of in vivo apoptosis and can be utilized to noninvasively assess the status of in vivo apoptosis in humans.

Keywords: Apoptosis; Biomarker; Fortilin; Programmed Cell Death.

Figures

Graphical abstract
Graphical abstract
Fig. 1
Fig. 1
Development and characterization of fortilin ELISA. Abbreviations: Ab, antibody; HRP, horseradish peroxidase; TMB,3,3′,5,5′-tetramethylbenzidine. A. The design of fortilin ELISA. B. Detection limits of the fortilin ELISA. Each span of the error bar represents 3 × standard deviation (SD). C. Histogram of mouse serum fortilin levels D. Probability plot of mouse serum fortilin levels using the normal distribution fit with the 5th and 95th percentile values of 26.84 and 69.48 ng/mL. E. Histogram of human serum fortilin levels. F. Probability plot of human serum fortilin levels using the gamma distribution fit with 5th and 95th percentile values of 18.68 and 163.55 ng/mL.
Fig. 2
Fig. 2
Serum fortilin levels are increased after anti-cancer therapy (radiation therapy, chemotherapy, or both). Abbreviations: *, statistically significant (P < 0.05); PRE, pre-anti-cancer therapy; POST, post-anti-cancer therapy. A. Change in serum fortilin levels [ng/mL] in 18 patients undergoing anti-cancer therapy. The details of the patients are in Table 1. B. Change in serum fortilin levels when pre-treatment levels were normalized to one. Patient #10: a sufficient amount of post-treatment sample was available only for fortilin ELISA, thus included for serum fortilin assay but excluded from the other assays. Patient #5: excluded from the study as post-treatment sample volume was not sufficient for any assays.
Fig. 3
Fig. 3
Infrequent increases in serum lactate dehydrogenase (LDH) levels suggest a lack of extensive necrotic tissue damage in the study patients. Abbreviations: LDH, lactate dehydrogenase; *, P < 0.05; PRE, pre-anti-cancer therapy; POST, post-anti-cancer therapy. A. Change in serum LDH levels when pre-treatment levels were normalized to one. B. Serum LDH levels did not correlate significantly with serum fortilin levels.
Fig. 4
Fig. 4
Known serum markers of apoptosis do not increase as much as fortilin levels upon anti-cancer therapy. Abbreviations: Cyt C; cytochrome c; n-DNA, nucleosomal DNA; fCK-18, fragmented cytokeratin-18; *, P < 0.05; PRE, pre-anti-cancer therapy; POST, post-anti-cancer therapy; Δ, fold change. A. Changes in serum cytochrome c (Cyt C) levels when their pre-treatment levels were normalized to one. There was no statistically significant association between serum Cyt C and fortilin levels by regression analysis (P = 0.635, R2 = 1.5%). B. Changes in serum nucleosomal DNA (n-DNA) levels when their pre-treatment levels were normalized to one. There was no statistically significant association between serum n-DNA and fortilin levels by regression analysis (P = 0.18, R2 = 11.9%). C. Changes in serum fragmented cytokeratin-18 (fCK-18) levels when their pre-treatment levels were normalized to one. There was no statistically significant association between serum fCK-18 and fortilin levels by regression analysis (P = 0.082, R2 = 18.8%).
Fig. 5
Fig. 5
Serum fortilin levels increase in response to Jo2-antibody-induced apoptosis in the liver. Abbreviations: PBS, phosphate-buffered saline; CTL, control; i.p., intraperitoneal injection; ALT, alanine aminotransferase; n-DNA, nucleosomal DNA; H&E, hematoxylin and eosin staining; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining; *, P < 0.05; **, P < 0.01; ***, P < 0.005. A. C57BL/6J mice were injected with Jo2 antibody and sacrificed 5–9 h later. B. The livers of control and Jo2-treated mice. C. Hematoxylin & eosin (H&E) and TUNEL staining of the control and Jo2-treated livers. Size bar: 100 μm. D. TUNEL index (%) of the control and Jo2-treated livers. E. Caspase-3 activities of control and Jo2-treated organs. F. Serum alanine aminotransferase (ALT) levels in control and Jo2-treated mice. G. Serum n-DNA levels in control and Jo2-treated mice. H. Serum fortilin levels in control and Jo2-treated mice.
Fig. 6
Fig. 6
Fortilin is released in the very early phase of apoptosis, before the compromise to the plasma membrane integrity. Abbreviations: α-Fas IgM, anti-human Fas IgM (clone CH11); CM, conditioned media; n-DNA, nucleosomal DNA; 7-AAD, 7-aminoactinomycin D; Cyt C, cytochrome c; fCK-18, fragmented cytokeratin-18; LDH, lactate dehydrogenase; PMΔ, plasma membrane change; *, P < 0.05. A. Experimental design. Jurkat cells were challenged by 12.5 ng/mL of anti-human Fas IgM (clone CH11) in Roswell Park Memorial Institute (RPMI) media with 5% fetal bovine serum (FBS). Cells and conditioned media were harvested at indicated time points and subjected to the respective sets of assays as described in the panel. 7-AAD cannot pass through the intact plasma membrane. 7-AAD cannot enter into the live cell. B. 7-AAD staining to detect cells with change in plasma membrane integrity. C. LDH concentrations in the conditioned media. D. n-DNA concentration in the cell lysates that reflect the progression of apoptosis occurring within the cell. E. Fortilin concentrations in the conditioned media. F. Cyt C levels in the conditioned media. G. n-DNA concentration in the conditioned media. H. fCK-18 concentration in the conditioned media.

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Source: PubMed

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