Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes

作者：Ishiguro Kimiko^*; Lin Z Ping; Penketh Philip G; Shyam Krishnamurthy; Zhu Rui; Baumann Raymond P; Zhu Yong Lian; Sartorelli Alan C; Rutherford Thomas J; Ratner Elena S

来源：Biochemical Pharmacology, 2014, 91(3): 312-322.

DOI：10.1016/j.bcp.2014.08.006

• Triapine (3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone)
• Dp44mT (di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone)
• Metal coordination
• DNA replication stress
• DNA double-strand breaks
• Reactive oxygen species

Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the alpha-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu2+ inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20 min that caused limited cell-kill, and the other occurring after 16 h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4 mu M caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16 h exposure to triapine paralleled the appearance of phospho-(gamma)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within I h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.

• 出版日期2014-10-1