title: Mechanism of apoptosis induced by a new topoisomerase inhibitor through the generation of hydrogen peroxide.
abstract: TAS-103, a new anticancer drug, induces DNA cleavage by inhibiting the activities of topoisomerases I and II. We investigated the mechanism of TAS-103-induced apoptosis in human cell lines. Pulsed field gel electrophoresis revealed that in the leukemia cell line HL-60 and the H(2)O(2)-resistant subclone, HP100, TAS-103 induced DNA cleavage to form 1-2-Mb fragments at 1 h to a similar extent, indicating that the DNA cleavage was induced independently of H(2)O(2). TAS-103-induced DNA ladder formation in HP100 cells was delayed compared with that seen at 4 h in HL-60 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded increases in mitochondrial membrane potential (DeltaPsim) and caspase-3 activation. Inhibitors of poly(ADP-ribose) polymerase (PARP) prevented both TAS-103-induced H(2)O(2) generation and DNA ladder formation. The levels of NAD(+), a PARP substrate, were significantly decreased in HL-60 cells after a 3-h incubation with TAS-103. The decreases in NAD(+) levels preceded both increases in DeltaPsim and DNA ladder formation. Inhibitors of NAD(P)H oxidase prevented TAS-103-induced apoptosis, suggesting that NAD(P)H oxidase is the primary enzyme mediating H(2)O(2) formation. Expression of the antiapoptotic protein, Bcl-2, in BJAB cells drastically inhibited TAS-103-induced apoptosis, confirming that H(2)O(2) generation occurs upstream of mitochondrial permeability transition. Therefore, these findings indicate that DNA cleavage by TAS-103 induces PARP hyperactivation and subsequent NAD(+) depletion, followed by the activation of NAD(P)H oxidase. This enzyme mediates O(2)(-)-derived H(2)O(2) generation, followed by the increase in DeltaPsim and subsequent caspase-3 activation, leading to apoptosis.
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University School of Medicine, Edobashi, Tsu, Mie 514-8507, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Mizutani
authors.forename: Saeko
authors.initials: S
authors.lastname: Tada-Oikawa
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Hiraku
authors.forename: Shinji
authors.initials: S
authors.lastname: Oikawa
authors.forename: Michio
authors.initials: M
authors.lastname: Kojima
authors.forename: Shosuke
authors.initials: S
authors.lastname: Kawanishi
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D004249:DNA Damage; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007192:Indenes / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000502:physiology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D059004:Topoisomerase I Inhibitors
chemical_list: D000634:Aminoquinolines; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D007192:Indenes; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D059004:Topoisomerase I Inhibitors; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D011065:Poly(ADP-ribose) Polymerases; C106647:6-((2-(dimethylamino)ethyl)amino)-3-hydroxy-7H-indeno(2,1-c)quinolin-7-one