Theranostics 2013; 3(3):190-200. doi:10.7150/thno.5825

Research Paper

Longitudinal Bioluminescence Imaging of the Dynamics of Doxorubicin Induced Apoptosis

Gang Niu1, Lei Zhu1, 2, Don N. Ho1, Fan Zhang1, 2, Haokao Gao1, Qimeng Quan1, Naoki Hida1, Takeaki Ozawa3, Gang Liu2✉, Xiaoyuan Chen1

1. Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892, USA.
2. Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, Fujian, 361002, China.
3. Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

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 GloTM 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.

Keywords: apoptosis, cyclic firefly luciferase, bioluminescence imaging, doxorubicin, caspase-3.

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How to cite this article:
Niu G, Zhu L, Ho DN, Zhang F, Gao H, Quan Q, Hida N, Ozawa T, Liu G, Chen X. Longitudinal Bioluminescence Imaging of the Dynamics of Doxorubicin Induced Apoptosis. Theranostics 2013; 3(3):190-200. doi:10.7150/thno.5825. Available from http://www.thno.org/v03p0190.htm