Abstract
Purpose. The aim of this paper is to develop new optical bioprobes for the imaging of apoptosis. Procedure. We developed quenched near-infrared probes which become fluorescent upon cleavage by caspase-3, the key regulatory enzyme of apoptosis. Results. Probes were shown to be selectively cleaved by recombinant caspase-3. Apoptosis of cultured endothelial cells was associated with an increased fluorescent signal for the cleaved probes, which colocalized with caspase-3 and was reduced by the addition of a caspase-3 inhibitor. Flow cytometry demonstrated a similar profile between the cleaved probes and annexin V. Ex vivo experiments showed that sections of hearts obtained from mice treated with the proapoptotic drug doxorubicin displayed an increase in the fluorescent signal for the cleaved probes, which was reduced by a caspase-3 inhibitor. Conclusion. We demonstrated the capacity of these novel probes to detect apoptosis by optical imaging in vitro and ex vivo.
Highlights
Apoptosis, or programmed cell death, is a well-organized mechanism that allows cells to die without leading to inflammation, contrary to necrosis [1, 2]
Apoptosis of cultured endothelial cells was associated with an increased fluorescent signal for the cleaved probes, which colocalized with caspase-3 and was reduced by the addition of a caspase3 inhibitor
Ex vivo experiments showed that sections of hearts obtained from mice treated with the proapoptotic drug doxorubicin displayed an increase in the fluorescent signal for the cleaved probes, which was reduced by a caspase-3 inhibitor
Summary
Programmed cell death, is a well-organized mechanism that allows cells to die without leading to inflammation, contrary to necrosis [1, 2]. Apoptosis plays a critical role in a number of physiological functions such as fetal development or tissue homeostasis regulation [3]. In addition to these physiological roles, considerable evidence suggests that changes in apoptosis play a major role in the development of various diseases. In cancer, inhibition of apoptosis leads to anarchic division of cells and favors tumor development. Many methods have been developed to detect apoptosis, most of them targeting phosphatidylserine (PS) which is normally expressed on the inner plasma membrane. In apoptotic conditions, aminophospholipid translocase inhibition and scramblase activation lead to PS translocation to the outer leaflet of the plasma membrane [5, 6]. Natural ligands of this phospholipid, annexin V or the C2A domain of synaptotagmin [8], have been labeled with radioisotopes (99mTc) for single
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