Radiolabeled isatin binding to caspase-3 activation induced by anti-Fas antibody.
BACKGROUND
Noninvasive imaging methods that can distinguish apoptosis from necrosis may be useful in furthering our understanding of diseases characterized by apoptotic dysregulation as well as aiding drug development targeting apoptotic pathways. We evaluated the ability of radiolabeled isatins to quantify caspase-3 activity induced by the activation of the extrinsic apoptotic pathway by the anti-Fas antibody in mice.


METHODS
The behavior of three different radiolabeled isatins ([(18)F]WC-II-89, [(18)F]WC-IV-3 and [(11)C]WC-98) was characterized in mice with and without anti-Fas antibody treatment by microPET imaging and biodistribution studies. The activity of [(18)F]WC-II-89 was also compared with [(99m)Tc]mebrofenin. The effect of pan-caspase inhibition with quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh) on [(18)F]WC-II-89 uptake was studied. Caspase-3 activity was confirmed by a fluorometric enzyme assay.


RESULTS
All three tracers behaved similarly in microPET and biodistribution studies. Increased retention of all tracers was observed in the livers of treated animals and several other organs, all of which demonstrated increased caspase-3 enzyme activity; however, impaired hepatobiliary excretion made attribution of these findings to caspase-3 activity difficult. The isatin [(18)F]WC-II-89 was retained at statistically significantly higher levels in the organs after anti-Fas antibody treatment while [(99m)Tc]mebrofenin activity cleared, suggesting specific binding to activated caspase-3, but the magnitude of increased binding was still relatively low. Caspase inhibition with Q-VD-OPh partially blocked [(18)F]WC-II-89 retention but completely blocked caspase-3 enzyme activity in the liver.


CONCLUSIONS
The radiolabeled isatins appear to bind specifically to caspase-3 in vivo, but their sensitivity is limited. Further optimization is required for these tracers to be useful for clinical applications.
Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA. chend@mir.wustl.edu
Delphine L
DL
Chen
Dong
D
Zhou
Wenhua
W
Chu
Phillip
P
Herrbrich
Jacquelyn T
JT
Engle
Elizabeth
E
Griffin
Lynne A
LA
Jones
Justin M
JM
Rothfuss
Marco
M
Geraci
Richard S
RS
Hotchkiss
Robert H
RH
Mach
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived; D017209:Apoptosis / Q000187:drug effects*; D002250:Carbon Radioisotopes / Q000493:pharmacokinetics; D053148:Caspase 3 / Q000378:metabolism*; D061945:Caspase Inhibitors; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005462:Fluorine Radioisotopes / Q000493:pharmacokinetics; D007510:Isatin / Q000493:pharmacokinetics*; D008099:Liver / Q000000981:diagnostic imaging / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D049268:Positron-Emission Tomography / Q000379:methods; D011485:Protein Binding; D011804:Quinolines / Q000494:pharmacology; D017556:Technetium Compounds / Q000493:pharmacokinetics; D014018:Tissue Distribution
D000590:Amino Acid Chloromethyl Ketones; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D002250:Carbon Radioisotopes; D061945:Caspase Inhibitors; D005462:Fluorine Radioisotopes; D011804:Quinolines; D017556:Technetium Compounds; C411023:anti-Fas monoclonal antibody; C468548:quinoline-val-asp(OMe)-CH2-OPH; D007510:Isatin; D053148:Caspase 3