Investigation of brain tumors using (18)F-fluorobutyl ethacrynic amide and its metabolite with positron emission tomography

Abstract

To date, imaging of malignant glioma remains challenging. In positron emission tomography-related diagnostic imaging, differential tumor uptake of 3'-deoxy-3'-[(18)F] fluorothymidine ([(18)F]FLT) has been shown to reflect the levels of cell proliferation and DNA synthesis. However, additional biomarkers for tumors are urgently required. Aberrant levels of glutathione transferase (GST) activity have been hypothesized to constitute such a novel diagnostic marker. Here, a C6 rat glioma tumor model was used to assess the ability of the positron emission tomography tracers, [(18)F]FLT and (18)F-fluorobutyl ethacrynic amide ([(18)F]FBuEA), to indicate reactive oxygen species-induced stress responses as well as detoxification-related processes in tumors. Using a GST activity assay, we were able to demonstrate that FBuEA is more readily catalyzed by GST-π than by GST-α. Furthermore, we showed that FBuEA-GS, a metabolite of FBuEA, elicits greater cytotoxicity in tumor cells than in normal fibroblast cells. Finally, in vitro and in vivo investigation of radiotracer distribution of [(18)F]FBuEA and [(18)F] FBuEA-GS revealed preferential accumulation in C6 glioma tumor cells over normal fibroblast cells for [(18)F]FBuEA-GS but not for [(18)F]FBuEA.

Keywords: C6-glioma; GST-α; fluorine-18; glutathione transferase; molecular imaging.

Figure 1

Structures of fluorine-18-labeled positron emission tomography tracers, ie, [¹⁸F]FLT, [¹⁸F]FBuEA, and [¹⁸F]FBuEA-GS. Abbreviations: FLT, fluorothymidine; FBuEA, fluorobutyl ethacrynic amide; FBuEA-GS, metabolite of fluorobutyl ethacrynic amide.

Figure 2

Schematic diagram depicting the role of PET tracers [¹⁸F]FLT, [¹⁸F]FBuEA, and [¹⁸F]FBuEA-GS in antioxidant-related and detoxification-related imaging processes. Abbreviations: FLT, fluorothymidine; FBuEA, fluorobutyl ethacrynic amide; FBuEA-GS, metabolite of fluorobutyl ethacrynic amide; GSH, glutathione; PET, positron emission tomography; ROS, reactive oxygen species; GST, glutathione transferase.

Figure 3

Effects of test compounds on GST-catalyzed GSH conjugation in the presence of GSTA1. IC₅₀ values for inhibition of formation of the CDNB–GSH conjugate for ethacrynic acid =1.1 μM (5 μM) and FBuEA =17.5 μM. Abbreviations: CDNB, chlorodinitrobenzene; FBuEA, fluorobutyl ethacrynic amide; GSH, glutathione; GST, glutathione transferase; GSTA1, glutathione S-transferase alpha-1; IC₅₀, half maximal inhibitory concentration.

Figure 4

Effects of test compounds on GST-catalyzed conjugation of GSH in the presence of GSTP1. IC₅₀ values for inhibition of CDNB–GSH conjugate formation for ethacrynic acid =0.5469 μM (4 μM) and FBuEA =6.018 μM. Abbreviations: CDNB, chlorodinitrobenzene; FBuEA, fluorobutyl ethacrynic amide; GSH, glutathione; GST, glutathione transferase; GSTP1, glutathione S-transferase Pi-1; IC₅₀, half maximal inhibitory concentration.

Figure 5

Cytotoxicity of FBuEA and its metabolite FBuEA-GS in C6 glioma and normal fibroblast cells. An MTT assay was performed to assess the percentage survival of cells treated with the various reagents. Abbreviations: DMSO, dimethyl sulfoxide; FBuEA, fluorobutyl ethacrynic amide; FBuEA-GS, metabolite of fluorobutyl ethacrynic amide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; h, hours.

Figure 6

Cellular uptake of [¹⁸F]FLT in C6 glioma cells (red) and normal fibroblast cells (blue). The radiolabeled fluorocompound was added to the cell plates for equilibrium followed by washing the medium to count the percentage residual radioactivity. Abbreviation: FLT, fluorothymidine.

Figure 7

Medical diagnostic imaging in rats using MRI and PET. Arrow heads indicate the tumor loci. Notes: (A) MRI images of a rat inoculated with a brain tumor on the right upper side. Coronal slices at various depths were generated. (B) Dynamic PET imaging collated over a period of 0–60 minutes for [¹⁸F]FLT in a C6 glioma-bearing rat injected with 2 mCi/0.45 mL doses of [¹⁸F]FLT. (C) Dynamic PET imaging of a control rat injected with 2.06 mCi/0.6 mL [¹⁸F]FLT. Abbreviations: FLT, fluorothymidine; MRI, magnetic resonance imaging; PET, positron emission tomography.

Figure 8

Dynamic PET images of [¹⁸F]FBuEA in (A) healthy rat and a (B) syngeneic C6 glioma-bearing rat. Images were acquired by summation of uptakes from 0 to 60 minutes post-injection. The injection doses were 0.5 mCi/0.6 mL and 0.617 mCi/0.6 mL, respectively. Coronal, transverse, and sagittal cross-sections correspond to lower left, upper left, and upper right images, respectively. (C) The brain was illustrated from three cross-sections: transverse, sagittal, and coronal. Abbreviations: FBuEA, fluorobutyl ethacrynic amide; PET, positron emission tomography.