Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide

• Published in: Life sciences (1973) Vol. 76; no. 13; pp. 1439 - 1453
• Main Authors: Mizutani, Hideki, Tada-Oikawa, Saeko, Hiraku, Yusuke, Kojima, Michio, Kawanishi, Shosuke
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Inc 11.02.2005
• Subjects: 8-oxo-7,8-dihydro-2′-deoxyguanosine; Aminoquinolines - pharmacology; Anthracyclines - pharmacology; Antibiotics, Antineoplastic - pharmacology; Apoptosis; Apoptosis - drug effects; Caspase 3; Caspases - metabolism; Chromatography, High Pressure Liquid; Deoxyguanosine - analogs & derivatives; Deoxyguanosine - pharmacology; DNA - biosynthesis; DNA - genetics; DNA Damage - drug effects; Doxorubicin; Doxorubicin - pharmacology; Electrochemistry; Enzyme Inhibitors - pharmacology; Flow Cytometry; HL-60 Cells; Humans; Hydrogen peroxide; Hydrogen Peroxide - metabolism; Indenes - pharmacology; Membrane Potentials - drug effects; NADPH Oxidases - antagonists & inhibitors; Oxidative DNA damage; Poly(ADP-ribose) Polymerase Inhibitors; Topoisomerase I Inhibitors; 8-oxo-7,8-dihydro-2′-deoxyguanosine; Hydrogen peroxide; Doxorubicin; Apoptosis; Oxidative DNA damage
• ISSN: 0024-3205; 1879-0631
• DOI: 10.1016/j.lfs.2004.05.040

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 2O 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 2O 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 2O 2-mediated pathways in apoptosis. Flow cytometry revealed that H 2O 2 formation preceded the increase in ΔΨm 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 2O 2 generation, although DOX-induced apoptosis may involve topoisomerase II inhibition. This oxidative DNA damage causes indirect H 2O 2 generation through PARP and NAD(P)H oxidase activation, leading to the ΔΨm increase and subsequent caspase-3 activation in DOX-induced apoptosis.