title: Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide.
abstract: The main anticancer action of doxorubicin (DOX) is believed to be due to topoisomerase II inhibition and free radical generation. Our previous study has demonstrated that TAS-103, a topoisomerase inhibitor, induces apoptosis through DNA cleavage and subsequent H(2)O(2) generation mediated by NAD(P)H oxidase activation [H. Mizutani et al. J. Biol. Chem. 277 (2002) 30684-30689]. Therefore, to clarify whether DOX functions as an anticancer drug through the same mechanism or not, we investigated the mechanism of apoptosis induced by DOX in the human leukemia cell line HL-60 and the H(2)O(2)-resistant sub-clone, HP100. DOX-induced DNA ladder formation could be detected in HL-60 cells after a 7 h incubation, whereas it could not be detected under the same condition in HP100 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded the increase in Delta Psi m and caspase-3 activation. Poly(ADP-ribose) polymerase (PARP) and NAD(P)H oxidase inhibitors prevented DOX-induced DNA ladder formation in HL-60 cells. Moreover, DOX significantly induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, in HL-60 cells at 1 h, but not in HP100 cells. DOX-induced apoptosis was mainly initiated by oxidative DNA damage in comparison with the ability of other topoisomerase inhibitors (TAS-103, amrubicin and amrubicinol) to cause DNA cleavage and apoptosis. These results suggest that the critical apoptotic trigger of DOX is considered to be oxidative DNA damage by the DOX-induced direct H(2)O(2) generation, although DOX-induced apoptosis may involve topoisomerase II inhibition. This oxidative DNA damage causes indirect H(2)O(2) generation through PARP and NAD(P)H oxidase activation, leading to the Delta Psi m increase and subsequent caspase-3 activation in DOX-induced apoptosis.
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University School of Medicine, 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: Michio
authors.initials: M
authors.lastname: Kojima
authors.forename: Shosuke
authors.initials: S
authors.lastname: Kawanishi
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000634:Aminoquinolines / Q000494:pharmacology; D018943:Anthracyclines / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology*; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007192:Indenes / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D059004:Topoisomerase I Inhibitors
chemical_list: D000634:Aminoquinolines; D018943:Anthracyclines; D000903:Antibiotics, Antineoplastic; D004791:Enzyme Inhibitors; D007192:Indenes; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D059004:Topoisomerase I Inhibitors; D004317:Doxorubicin; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; C055866:amrubicin; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003849:Deoxyguanosine; C106647:6-((2-(dimethylamino)ethyl)amino)-3-hydroxy-7H-indeno(2,1-c)quinolin-7-one