A fluorescence anisotropy‐based assay to determine the activity of tissue transglutaminase in human cancer cell lines

High activity of tissue transglutaminase (TGase 2) in various tumors is associated with both their increased metastatic and invasive potential and their resistance towards chemotherapy and radiation. This renders TGase 2 an attractive target for the development of agents that are capable of targeting the tumor-associated TGase 2 for both imaging and therapeutic approaches [1].
To identify Inhibitor-based compounds for these purposes, the establishment of activity assays that allow their characterization both in vitro and at the cellular level is essential. We previously reported a kinetic fluorescence anisotropy (FA)-based assay to determine the transamidase activity of guinea pig TGase 2 [2] which follows the incorporation of fluorescently labeled (either fluoresceine or rhodamine B) cadaverine derivatives into N,N-dimethylated casein (DMC) over time. Using this assay, we were able to show that the method of FA ensures the absence of background signal and a high reproducibility in a homogenous assay design.
Here we apply the FA assay using DMC and a newly developed rhodamine B-cadaverine conjugate to recombinant human TGase 2 for the kinetic characterization of selected inhibitors and the active-site titration of the enzyme. Due to the favorable signal-to-noise ratio, it was furthermore possible to apply the FA assay for determining cellular TGase 2 in 25 human cell models by measuring the enzyme activity in the whole cell lysate and calculating the respective protein amount. The obtained results were compared with those of a densitometric Western Blot analysis, showing a clear correlation between the two data sets. The human lung cancer cell line NCI-H292 and the human breast cancer cell line MDA-MB-231 were found to exhibit the highest amount of activatable TGase 2 among the tested cancer cell lines, whereas the human cerebral endothelial cell line hCMEC-D3 showed the highest activity among the tested non-cancerous cell lines. To prove that the observed FA signal is caused by TGase 2 activity, N2-phenylacetyl-N6-acryloyl-lysine-4-(6-methylpyridine-2-yl)piperazide [3], which has previously been characterized as irreversible inactivator of this enzyme, was utilized. Application of this inhibitor resulted in a substantial reduction of the FA signal.

References:

[1] Pietsch et al. Bioorg. Med. Chem. Lett. 2013, 23, 6528.
[2] Hauser et al. Amino Acids 2016 DOI: 10.1007/s00726-00016-02192-00725.
[3] Wityak et al. ACS Med. Chem. Lett. 2012, 3, 1024.