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 H₂O₂ 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 H₂O₂-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 H₂O₂-mediated pathways in apoptosis. Flow cytometry revealed that H₂O₂ 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 H₂O₂ generation, although DOX-induced apoptosis may involve topoisomerase II inhibition. This oxidative DNA damage causes indirect H₂O₂ generation through PARP and NAD(P)H oxidase activation, leading to the ΔΨm increase and subsequent caspase-3 activation in DOX-induced apoptosis.

Introduction

Doxorubicin (DOX), an anthracycline antibiotic, is one of the most popular anticancer drugs. DOX is used for the treatment of human cancers including a variety of solid cancers (Chabner et al., 1996). Although the main anticancer action of DOX is believed to involve DNA damage through topoisomerase II inhibition and free radical generation by redox reaction (Chabner et al., 1996, Gewirtz, 1999, Jung and Reszka, 2001, Hurley, 2002), the mechanism of apoptosis induced by DOX has not well been clarified.

A number of anticancer drugs including DOX exert their effects by inducing apoptosis (Sellers and Fisher, 1999, Kaufmann and Earnshaw, 2000). The major apoptotic pathway of anticancer drugs acts through the loss of mitochondrial membrane integrity (Kaufmann and Earnshaw, 2000). A sudden increase in mitochondrial membrane permeability, so-called mitochondrial permeability transition (MPT), is a central coordination event in the apoptotic process (Mignotte and Vayssiere, 1998, Costantini et al., 2000, Gottlieb, 2000, Kroemer and Reed, 2000). MPT causes the release of cytochrome c from mitochondria; cytochrome c then activates effector caspases to induce DNA ladder formation. Reactive oxygen species (ROS), such as H₂O₂ and O₂⁻, cause apoptosis through MPT (Kroemer et al., 1997, Hampton et al., 1998, Chandra et al., 2000); some anticancer drugs induce ROS formation in apoptosis (Gewirtz, 1999, Muller et al., 1998, Ikeda et al., 1999, Tada-Oikawa et al., 1999, Kajiwara et al., 2001, Mizutani et al., 2002, Nakagawa et al., 2002, Tsang et al., 2003), and we have recently reported that TAS-103, a topoisomerase inhibitor and not a redox agent, induces apoptosis through indirect H₂O₂ generation (Mizutani et al., 2002). However, the mechanism of ROS formation by anticancer drugs including DOX remains to be clarified.

To clarify the mechanism of DOX-induced apoptosis, we investigated apoptosis by examining DNA ladder formation, H₂O₂ generation, mitochondrial membrane potential (ΔΨm) change and caspase-3 activation in HL-60 cells and HP100 cells, H₂O₂-resistant cells derived from HL-60. The mechanism of apoptosis was also examined by using inhibitors of poly(ADP-ribose) polymerase (PARP) and NAD(P)H oxidase. Moreover, in order to clarify whether H₂O₂ generation by DOX induces DNA damage, we measured the content of 8-oxo-7,8- dihydro-2′-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage, in HL-60 and HP100 cells treated with DOX using by high-performance liquid chromatography with an electrochemical detector (HPLC-ECD). In addition, we compared the ability of DOX to cause DNA damage and DNA ladder formation with that of topoisomerase inhibitors [TAS-103, amrubicin (AMR) and amrubicinol (AMR-OH), an active metabolite of AMR]. AMR is a new, completely synthetic 9-aminoanthracycline derivative which induces anticancer effects by stabilizing topoisomerase II-DNA complex (Hanada et al., 1998).