Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide
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).
Section snippets
Materials
DOX was purchased from Wako Pure Chemical Industries (Osaka, Japan). TAS-103 was provided by Taiho Pharmaceutical Co., Ltd (Tokyo, Japan). AMR and AMR-OH were provided by Sumitomo Pharmaceutical Co., Ltd (Osaka, Japan). Proteinase K was obtained from Merck (Darmstadt, Germany). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) and 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)] were purchased from Molecular Probes (Eugene, OR). z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC) and
DNA ladder formation in HL-60 and HP100 cells treated with DOX
We have analyzed DNA ladder formation, which is characteristic for apoptosis, in the cells treated with DOX using conventional electrophoresis. The DNA ladder formation could be detected at 1-5 μM in HL-60 cells after an 8 h incubation, whereas it could be detected apparently at 2–5 μM in HP-100 cells. (Fig. 1A). The time-course of the DNA ladder formation by 1 μM DOX was shown in Fig. 1B. It was detectable at 7 h in HL-60 cells, whereas it was observed slightly at 8 h in HP100 cells. The cell
Discussion
The present study showed that DOX induced apoptosis in both HL-60 cells and the H2O2-resistant clone, HP100 cells. The apparent DNA ladder formation could be detected at above 1 μM in HL-60 cells after an 8 h incubation, whereas it could be detected at above 2 μM in HP-100 cells. In HP100 cells, DNA ladder formation, H2O2 generation, ΔΨm increase and caspase-3 activation were delayed, compared with those of HL-60 cells. H2O2 formation preceded the ΔΨm increase and caspase-3 activation in
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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