Abstract
Apoptosis has a critical role in both physiological and pathological processes, and therefore probes that enable direct and fast visualization for apoptosis in vitro and in vivo have great significance for evaluation of therapeutic effects, disease monitoring and drug screening. We report here a novel apoptotic marker heat shock protein 60 (HSP60)-based apoptosis imaging probe, P17. In this study, we show that P17 can label multiple drug-induced apoptotic cells in vitro, and the difference in binding intensities between apoptotic and viable cells by fluorescent P17 is more than 10-fold in six cell lines measured by flow cytometry and proportional to the apoptotic level of the cells. We further visualized the apoptosis in the subcutaneous tumor of mice by vein injection of P17 using in vivo fluorescent imaging. P17 was identified to bind specifically to HSP60 accumulated in apoptotic cells by pull-down experiments and mass spectrometry. Furthermore, the P17 binding was correlated with the apoptotic feature of phosphatidylserine (PS) exposure and caspase-3 activation. We also clarify that P17 labels the cells in late stage apoptosis by double staining with different stage markers, unveiling that HSP60 may be involved with late stage of apoptosis. Overall, this study has demonstrated that P17 is a novel apoptosis probe targeting HSP60 and promising for the detection of apoptosis in vitro and in vivo.
Highlights
Apoptosis is a critical biological process, occurring normally during development and aging,[1] and having important roles in pathological processes
We showed that P17 was likely to label the apoptotic cells in the late stage, unveiling heat shock protein 60 (HSP60) was closely involved with late apoptosis
Despite the complexity of the roles HSP60 has in apoptosis process, data exist supporting an apparent accumulation of HSP60 in the cytoplasm of Jurkat,[10,36] Hela,[38] MDA-MB-23114 and cardiac myocytes[37] during apoptosis
Summary
Apoptosis has a critical role in both physiological and pathological processes, and probes that enable direct and fast visualization for apoptosis in vitro and in vivo have great significance for evaluation of therapeutic effects, disease monitoring and drug screening. We report here a novel apoptotic marker heat shock protein 60 (HSP60)-based apoptosis imaging probe, P17. We show that P17 can label multiple drug-induced apoptotic cells in vitro, and the difference in binding intensities between apoptotic and viable cells by fluorescent P17 is more than 10-fold in six cell lines measured by flow cytometry and proportional to the apoptotic level of the cells. We clarify that P17 labels the cells in late stage apoptosis by double staining with different stage markers, unveiling that HSP60 may be involved with late stage of apoptosis. This study has demonstrated that P17 is a novel apoptosis probe targeting HSP60 and promising for the detection of apoptosis in vitro and in vivo. Oncogenesis (2016) 5, e201; doi:10.1038/oncsis.2016.14; published online 29 February 2016
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