| Objective:Apoptosis is a critical biological process, not only occurring normally duringdevelopment and aging, but also playing important roles in pathological processes. For examples, too much loss of cardiomyocytes and neurons may lead to cardiovascular and neurodegenerative diseases respectively; while too littleapoptosis can give rise to cancer, autoimmune diseases and other morbidities. Therefore, apoptosis has been developed as one of the important therapeutic strategies. Thus, identifying cell apoptosis directly and rapidly is of great significance to monitoring and assessing the effectiveness or toxic side effects of drugs or therapies as well as to further understanding the mechanism of apoptosis and the pathogenesis of apoptosis-involved diseases. As the most widely used in vitro apoptosis detector, Annexin V, has been limited for further clinical use because of its suboptimal pharmacokinetics, apoptosis probes to newtargets and with low molecular weight are keenly needed both in basic research andin clinical applications.Considering the advantages of designed peptides, such as low cost and flexiblemodification,we screened one peptide sequence which can bind to apoptotic cells efficiently, from a collection of chemically synthesized peptides based on self-assembly technology. To further explore the apoptosis-imaging potential of the peptide, we testedpeptide in vitroin various cell lines and also in vivo utilizing different apoptosis-inducing stimuli. We also identified and verified the targeting protein of the peptide in a protein-protein interaction study. Furthermore, we evaluated the safety of the peptide in vitro and in healthy mice in vivo.Methods:In TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptotic Jurkat cell model, Annexin V and cleaved caspase-3 antibody were used for colocalization assay to prove the efficacy of peptide in revealing apoptotic cells by flow cytometry and confocal imaging, and the concentration, incubation time and apoptosis degree effects of peptide wereexamined by flow cytometry. To characterize the preference of peptide to apoptotic cells in different stages, double staining assays with early or late apoptotic markers were performed using flow cytometry. The binding partners of peptide were captured by pull-down assay andidentified by MALDI-FTICR, followed by verification using colocalization analysis and western blot. By four different methods, including flow cytometry, fluorescence microscopy, confocal imaging and microplate reader, the potencyof peptide was tested in multiple apoptotic cell lines. Fluorescent peptide was also intravenously injected to detect apoptosis in a 4T1 tumor- bearing mice model and a doxorubicin-induced acute cardiotoxicity mice model, and the apoptosis analysis of tissue sections was performed by TUNEL and cleaved caspase-3 staining. The distribution and clearance of peptide in vivo were investigated in healthy BALB/c mice using ex vivo imaging. The effects of peptide on cell viability and apoptosis were tested by CCK-8 and Annexin V/PI staining, and the effects on normal cell functions, such as cytoskeleton and adhesion, were performed by confocal microscopy and adhesion assay. The safety of peptide in vivo was evaluated in healthy BALB/c mice with intravenous injection, using histological analysis of the tissue sections from normal organs with the highest accumulation of peptide.Results:(1) Screened water-soluble peptide P17 can label multiple drug-induced apoptotic cells, including human leukemia cells, human solid tumor cells, human vascular endothelial cell, mouse primary myocardial cell and rat myocardial cell. (2) P17 has high affinity to apoptotic cells with a fluorescent signal enhancement of at least 10-fold greater than that of the viablecontrol; The binding platform of P17 was reachedafter 15 min incubation and maintained to 4 h, demonstrating the rapid and stable binding of P17 to apoptotic cells. (3) Double-staining with early or late apoptosis markers, P17 showed notable prominence to label cells in latestage of apoptosis. (4) The P17-captured protein in pull-down assay was identified to be Heat Shock Protein 60 (HSP60). Colocalization analysis and western blot with HSP60 antibody showed that P17 specifically bound to HSP60 in thecytoplasm and HSP60 apparently accumulated in apoptotic cells. (5) In 4T1 tumor-bearing mice model, P17showed a significant accumulation in the tumor mass compared to vehiclegroup, and the TUNEL and cleaved caspase-3 staining confirmed that the fluorescencesignal that was observed by ex vivo imaging labeled the apoptotic region. (6) Within 1 to 9 hours after P17 had been injection, in doxorubicin-induced acute cardiotoxicity mice, the whole-body fluorescenceimaging andex vivoimaging of theexcised heart showed significantly higher fluorescence signals compared to vehicle group. And the fluorescent intensity in peripheral blooddecreased steadily, which were approximately back to control levels after 8 h. While in health mice, the bladder region showed significantly stronger signals than other tissues in whole-body imaging, and there was no visible signal in the excised hearts in ex vivo imaging. After 1 h injection of P17, the fluorescent intensity in peripheral blood were 143 times control, but thereafter decreased significantly, and after 8 h it was approximately back to control levels. (7) The fluorescent signal of P17 dropped to control level within 6 h in liverand kidney in healthy mouse model. (8) CCK-8 and Anneixn V/PI staining showed that P17 itself had no effects on cell viability and apoptosis. And No significant effects of P17 emerged on cell cytoskeleton and adhesion. (9) No degeneration, engorgement, congestion or infiltration of inflammatory cells was observed in the H&E staining tissue sections of liver and kidney, the two organs with the highest accumulation of P17 in healthy mice.Conclusion:Peptide P17 has the ability of labeling multiple kinds of apoptotic cells in vitro andapoptosis in vivo with rapid distribution and clearancethrough binding with HSP60 in cytoplasm, exhibiting promisingpotentials for uses in the visualization of apoptosis in the biomedical researches andapplications. This studyalso unveils that HSP60 isclosely involved with late apoptosis and offer a new perspective that would help to understand the mechanism of HSP60 during apoptosis. |
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