Abstract
The chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) shows potent and selective anticancer and antimetastatic activity. However, the mechanism by which it is initially transported into cells to induce cytotoxicity is unknown. Hence, the current investigation examined the cellular uptake of 14C-Dp44mT relative to two structurally related ligands, namely the aroylhydrazone 14C-pyridoxal isonicotinoyl hydrazone (14C-PIH) and the thiosemicarbazone 14C-2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (14C-Bp4eT). In marked contrast to the cellular uptake of 14C-PIH and 14C-Bp4eT, which were linear as a function of concentration, 14C-Dp44mT uptake was saturable using SK-N-MC neuroepithelioma cells (Bmax, 4.28 × 107 molecules of chelator/cell; and Kd, 2.45 μM). Together with the fact that 14C-Dp44mT uptake was temperature-dependent and significantly (P < 0.01) decreased by competing unlabeled Dp44mT, these observations indicated a saturable transport mechanism consistent with carrier/receptor–mediated transport. Other unlabeled ligands that shared the saturated N4 structural moiety with Dp44mT significantly (P < 0.01) inhibited 14C-Dp44mT uptake, illustrating its importance for carrier/receptor recognition. Nevertheless, unlabeled Dp44mT most markedly decreased 14C-Dp44mT uptake, demonstrating that the putative carrier/receptor shows high selectivity for Dp44mT. Interestingly, in contrast to 14C-Dp44mT, uptake of its Fe complex [Fe(14C-Dp44mT)2] was not saturable as a function of concentration and was much greater than the ligand alone, indicating an alternate mode of transport. Studies examining the tissue distribution of 14C-Dp44mT injected intravenously into a mouse tumor model demonstrated the 14C label was primarily identified in the excretory system. Collectively, these findings examining the mechanism of Dp44mT uptake and its distribution and excretion have clinical implications for its bioavailability and uptake in vivo.
Footnotes
- Received July 15, 2013.
- Accepted September 30, 2013.
D.S.K. and D.R.R. contributed equally to this work as cocorresponding senior authors.
This work was supported by the National Health and Medical Research Council of Australia [Project Grants No. 1021607, No. 1021601, and No. 1048972; Senior Principal Research Fellowship No. 571123 (to D.R.R. and D.S.K.)]; and Cancer Institute New South Wales [Early Career Development Fellowship 08/ECF/1-30]. A.M.M. acknowledged with sincere appreciation a Bob and Nancy Edwards Postgraduate Scholarship from the Sydney Medical School, University of Sydney. The authors declare no conflict of interest.
↵This article has supplemental material available at http://molpharm.aspetjournals.org.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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