title: 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.
abstract: Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-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 Cu(2+) 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 μM 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-(γ)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 1h 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.
authors.affiliation: Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Yale University School of Medicine, New Haven, CT 06520, United States. Electronic address: kimiko.ishiguro@yale.edu.
authors.forename: Kimiko
authors.initials: K
authors.lastname: Ishiguro
authors.affiliation: Department of Pharmacology and Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Z Ping
authors.initials: ZP
authors.lastname: Lin
authors.affiliation: Department of Pharmacology and Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Penketh
authors.affiliation: Department of Pharmacology and Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Krishnamurthy
authors.initials: K
authors.lastname: Shyam
authors.affiliation: Department of Pharmacology and Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhu
authors.affiliation: Department of Pharmacology and Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Raymond P
authors.initials: RP
authors.lastname: Baumann
authors.affiliation: Department of Pharmacology and Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Yong-Lian
authors.initials: YL
authors.lastname: Zhu
authors.affiliation: Department of Pharmacology and Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Alan C
authors.initials: AC
authors.lastname: Sartorelli
authors.affiliation: Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Rutherford
authors.affiliation: Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Yale University School of Medicine, New Haven, CT 06520, United States. Electronic address: elena.ratner@yale.edu.
authors.forename: Elena S
authors.initials: ES
authors.lastname: Ratner
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003300:Copper / Q000494:pharmacology*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004798:Enzymes / Q000378:metabolism; D018922:HL-60 Cells / Q000187:drug effects; D006657:Histones / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D011725:Pyridines / Q000494:pharmacology*; D013882:Thiosemicarbazones / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D000970:Antineoplastic Agents; D004798:Enzymes; C468783:H2AX protein, human; D006657:Histones; D011725:Pyridines; D013882:Thiosemicarbazones; C539263:di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone; C078157:3-aminopyridine-2-carboxaldehyde thiosemicarbazone; C013996:2-formylpyridine thiosemicarbazone; D003300:Copper
keywords: DNA double-strand breaks; DNA replication stress; Dp44mT (di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone); Metal coordination; Reactive oxygen species; Triapine (3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone)