Distinct mechanisms of site‐specific oxidative DNA damage by doxorubicin in the presence of copper(II) and NADPH‐cytochrome P450 reductase

Abstract

The anticancer mechanism of doxorubicin (DOX), an anthracycline antibiotic, is believed to involve DNA damage through topoisomerase II inhibition and free radical generation. The free radical generation may also participate in genotoxicity, as well as cardiotoxicity, in normal human cells. The present study showed that DOX generates 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), an indicator of oxidative DNA damage, in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. Since DOX has both p-quinone and p-hydroquinone residues, free radical generation can be initiated by either reduction or oxidation of DOX. To clarify whether the oxidized or reduced form is more important for DOX-induced H(2)O(2) generation, we investigated the site-specific DNA damage induced by DOX in the presence of Cu(II), in comparison with that in the presence of cytochrome P450 reductase, using (32)P-labeled DNA fragments. DOX caused DNA damage in the presence of Cu(II) or cytochrome P450 reductase. The degree of Cu(II)-mediated DNA damage, including 8-oxodG formation, was much greater than that of cytochrome P450 reductase-mediated DNA damage. DOX plus Cu(II) caused DNA damage specifically at guanine, thymine and cytosine residues, particularly at 5'-GG-3', 5'-GT-3' and 5'-TG-3' sequences. Scavenger experiments suggested the involvement of reactive species generated from H(2)O(2) and Cu(I). When cytochrome P450 reductase and NADPH were used instead of Cu(II), every nucleotide was uniformly damaged, suggesting the participation of.OH. We conclude that DOX may induce carcinostatic and genotoxic effects through oxidation of its p-hydroquinone moiety by metal ion rather than through p-quinone reduction by cytochrome P450 reductase.

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

“…Apocynin (APO) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). 32 P-labeled DNA fragment was also obtained from the human c-Ha-ras-1 protooncogene (Mizutani et al, 2003).…”

Low-energy-loss electron microscopy of doxorubicin in human breast cancer MCF-7 cells: Localization by color

“…Although it has been studied extensively, these studies have not crystallized into a single unified idea of its mode of action. Biochemical studies point to intercalation of DNA (Goto et al, 2001), complexation with and inhibition of topoisomeraseII (D'Arpa D' Arpa and Liu, 1989) or to oxidative DNA damage by DOX-induced radical formation (Mizutani et al, 2003). Structural studies have implicated various subcellular structures such as the plasma membrane (Arancia, 1988;Arancia et al, 1995), the nuclear membrane and chromatin (Altan et al, 1998), and the nucleolus (Merski et al, 1976(Merski et al, , 1978Unverferth et al, 1983;Cavanagh et al, 1987;Abe et al, 1996).…”

Stimulatory Effects of Hydroxyl Radical Generation by Ga-Al-As Laser Irradiation on Mineralization Ability of Human Dental Pulp Cells

“…In the present study, to elucidate the mechanism of promotion of hard tissue formation by Ga-Al-As laser irradiation, we focused on · OH generation and investigated its effects on hard tissue formation by HDP cells. While it has been reported that · OH is involved in apoptosis, 26) inflammation, 27) DNA damage, 28) and aging, 29) it has also become clear that · OH also plays roles in cellular signal transduction. 30) Nishida et al reported 19) that activation of MAP kinase was dependent on activation of Gprotein when rat neonatal cardiomyocytes were treated with H 2 O 2 , indicating that · OH directly activates G-protein independently of receptors.…”