TAS-103 is
a DNA intercalating indeno-
quinoline derivative that stimulates DNA cleavage by topoisomerases. This
synthetic drug has a broad spectrum of antitumor activity against many human solid
tumor xenografts and is currently undergoing clinical trials. We investigated the induction of apoptosis in human promyelocytic
leukemia cells treated with
TAS-103. The treatment of proliferating human
leukemia cells for 24 h with various concentrations of the drug induces significant variations in the mitochondrial transmembrane potential (delta(psi)mt) measured by flow cytometry using the
fluorochromes 3,3-dihexyloxacarbocyanine
iodide, Mitotracker Red, and tetrachloro-tetraethylbenzimidazolcarbocyanine
iodide. The collapse of delta(psi)mt is accompanied by a marked decrease of the intracellular pH. Cleavage experiments with the substrates N-acetyl-
Asp-Glu-Val-Asp-pNA,
poly(ADP-ribose) polymerase, and
pro-caspase-3 reveal unambiguously that
caspase-3 is a key mediator of the apoptotic pathway induced by
TAS-103.
Caspase-8 is also cleaved, and the bcl-2
oncoprotein is underexpressed. Drug-induced internucleosomal DNA fragmentation and the externalization of
phosphatidylserine residues in the outer leaflet of the plasma membrane were also characterized. The cell cycle perturbations produced by
TAS-103 can be connected with the changes in deltapsi(mt). At low concentrations (2-25 nM), the drug induces a marked G2 arrest and concomitantly provokes an increase in the potential of mitochondrial membranes. In contrast, treatment of the HL-60 cells with higher drug concentrations (50 nM to 1 microM) triggers massive apoptosis and a collapse of deltaP(mt) that is a signature for the opening of the mitochondrial permeability transition pores. The discovery of a correlation between the G2 arrest and changes in mitochondrial membrane potential provides an important mechanistic insight into the action of
TAS-103.