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Longitudinal bioluminescence imaging of the dynamics of Doxorubicin induced apoptosis.

Niu G, Zhu L, Ho DN, Zhang F, Gao H, Quan Q, Hida N, Ozawa T, Liu G, Chen X. Theranostics. 2013;3(3):190-200.

Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892, USA.

 

Abstract

Objectives: Most chemotherapy agents cause tumor cell death primarily by the induction of apoptosis. The ability to noninvasively image apoptosis in vivo could dramatically benefit pre-clinical and clinical evaluation of chemotherapeutics targeting the apoptotic pathway. This study aims to visualize the dynamics of apoptotic process with temporal bioluminescence imaging (BLI) using an apoptosis specific bioluminescence reporter gene. Methods: Both UM-SCC-22B human head and neck squamous carcinoma cells and 4T1 murine breast cancer cells were genetically modified with a caspase-3 specific cyclic firefly luciferase reporter gene (pcFluc-DEVD). Apoptosis induced by different concentrations of doxorubicin in the transfected cells was evaluated by both annexin V staining and BLI. Longitudinal BLI was performed in xenografted tumor models at different time points after doxorubicin or Doxil treatment, to evaluate apoptosis. After imaging, DNA fragmentation in apoptotic cells was assessed in frozen tumor sections using TUNEL staining. Results: Dose- and time-dependent apoptosis induced by doxorubicin in pcFluc-DEVD transfected UM-SCC-22B and 4T1 cells was visualized and quantified by BLI. Caspase-3 activation was confirmed by both caspase activity assay and Glo(TM) luciferase assay. One dose of doxorubicin treatment induced a dramatic increase in BLI intensity as early as 24 h after treatment in 22B-pcFluc-DEVD xenografted tumors. Sustained signal increase was observed for the first 3 days and the fluorescent signal from ex vivo TUNEL staining was consistent with BLI imaging results. Long-term imaging revealed that BLI signal consistently increased and reached a maximum at around day 12 after the treatment with one dose of Doxil. Conclusions: BLI of apoptosis with pcFluc-DEVD as a reporter gene facilitates the determination of kinetics of the apoptotic process in a real-time manner, which provides a unique tool for drug development and therapy response monitoring.

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Additional Information

Gang Liu; Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, Fujian, 361002, China. Email: [email protected]

After confirming the sensitivity and specificity of the caspase-3 specific cyclic firefly luciferase reporter gene (pcFluc-DEVD) in vitro, the authors successfully visualized and quantified doxorubicin induced apoptosis in tumor cells and xenografted tumors longitudinally. Altogether, bioluminescence imaging of apoptosis with pcFluc-DEVD as a reporter gene facilitates the determination of kinetics of apoptosis process in a real-time manner, which provides a unique tool for drug development, therapy response monitoring, drug delivery and release and radionuclide imaging probe development.

Figure legend

The biosensor is composed of an engineered firefly luciferase, of which the N- and C-terminal ends are linked with DEVD, a substrate sequence of caspase-3. Upon caspase-3 activation during apoptosis, the DEVD sequence is cleaved and the cyclized luciferase recovers its activity. This biosensor can be used for imaging apoptosis induced by chemotherapeutics to monitor effects of cytotoxic compounds or novel pharmacological chemicals, as most cells activate caspase-3 during apoptosis. The cleavage site of this system can also be modified to “report” on the kinetics of a variety of proteolytic processes in vitro and in vivo by using bioluminescence imaging.

 

Longitudinal Bioluminescence Imaging of the Dynamics of Doxorubicin Induced Apoptosis. Global Medical Discovery