Mode of action of copper complexes of thiosemicarbazones in human leukemia HL-60 cells

Copper complexes of thiosemicarbazones are potent cytotoxic agents against a wide variety of human and murine cancers. Previous reported studies in the literature have shown that the complexes cause DNA strand scission in L1210 cells, inhibit multiple enzymes involved in DNA synthesis and induce apoptosis in certain cell lines. Some of the copper complexes also demonstrated the ability to inhibit isolated DNA topoisomerase II activity in the low micromolar range.; The N4 substituted 2-acetylpyrazine (CCT 1) and 2-acetylpyridine (CCT 2) thiosemicarbazone copper complexes were chosen to continue mode of action studies in human leukemia cells. CCTs 1 and 2 were shown to be potent cytotoxic agents that suppress DNA synthesis and induce DNA damage in HL-60 cells at 8 muM as early as 3 h. The CCTs induced apoptosis after 12 h incubation as demonstrated by cytochrome c release, PARP and ICAD cleavage and the activation of DNA endonucleases. CCTs elevated caspase 2 activity and may activate an alternate stress pathway to induce apoptosis at an early time of 3 h. Although the CCTs induced apoptosis, it was unclear if apoptosic events were responsible for the early activation of a DNA endonuclease and DNA damage in HL-60 cells. Experiments conducted in HL-60 cells and a HL-60 clone that expresses lowered amounts of DNA topoisomerase II enzyme demonstrated that the CCTs did inhibit DNA topoisomerase II activity intracellularly.; The CCTs also inhibited isolated human DNA topoisomerase II activity in a similar manner as in HL-60 cells. Experiments in this system demonstrated that the CCTs inhibited DNA topoisomerase II activity in a manner that does not produce protein linked DNA breaks like etoposide, a clinically useful anti-cancer agent that targets DNA topoisomerase II. The CCTs inhibited the ATPase activity in this system and may explain how the CCTs are exerting their effects. The ATPase activity may be inhibited by two distinct mechanisms in the catalytic cycle of DNA topoisomerase II (1) by blocking the binding of ATP to the enzyme or (2) by preventing the hydrolysis of ATP by the enzyme. The present experiments suggest that the CCTs are acting by the former.