Impact Of Antioxidants On Myeloperoxidase (Mpo)-Dependent Dna Damage And Genotoxicity Induced By Etoposide (Vp-16): Implications For Therapy-Induced Second Malignancies

Previous work (Mol. Pharm. 79: 479-87, 2011) demonstrated that MPO, found in myeloid progenitor cells, oxidized the anticancer agent VP-16 to its phenoxyl radical (VP-O•) and led to enhanced topoisomerase II-mediated strand cleavage through redox cycling resulting in MLL translocations, implicating MPO in VP-16 leukemogenesis. Utilizing shRNA MPO in myeloid leukemia HL60 cells, MPO dependency for VP-16 activity was further established (PAACR 55: 826, 2014). In the present study, we examined the effects of dehydroascorbate (DHA) and trolox on MPO-dependent effects of VP-16, VP-16 catechol (VP-OH) and VP-16 ortho-quinone (VP-oQ, fully oxidized VP-16). In addition, the role of GSH as a mediator of the pro- or anti-oxidant effects of VP-16, its metabolites and several other phenolic agents was evaluated. Using Comet assays, VP-16-induced DNA strand breaks in MPO+ HL60 cells were significantly reduced when cells were pre-incubated with 1 mM DHA; known to act as a reductant of MPO generated VP-O•. In MPO knockdowns, VP-16-induced DNA damage was diminished compared to MPO+ cells. In these MPO knockdowns, DHA did not perturb VP-16-induced DNA damage. Similar results were demonstrated with VP-OH, consistent with MPO-mediated generation of VP-O• and DHA reduction/protection against DNA damage. For VP-oQ, 1,4-benzoquinone, and camptothecin, DNA damage was similar in MPO+ and knockdown cells with no attenuation in the presence of DHA. VP-16 and VP-OH also induced oxidative DNA damage in MPO+ cells which was reduced by DHA. This DNA damage was attenuated in MPO knockdowns with no further effect by DHA. The phenolic vitamin E analog trolox, a known MPO substrate, also reduced oxidative DNA damage induced by VP-16 and VP-OH but not by VP-oQ. Using 3′-(p-hydroxyphenyl) fluorescein (HPF), pro-oxidant activity of VP-16 was demonstrated in MPO+ cells which converted to anti-oxidant effects in MPO knockdowns. Both DHA and trolox protected cells against VP-16-induced pro-oxidant effects in MPO+ cells. When GSH levels were reduced by incubation with buthionine sulfoximine, the pro-oxidant effects of VP-16 in MPO+ cells were eliminated. N-acetyl cysteine (NAC) restored VP-16-induced pro-oxidant activity. In contrast, the pro-oxidant effects of the phenolic agents quercetin and EGCG were found to be independent of MPO and were diminished by addition of NAC. Together, results strongly suggest that MPO-catalyzed oxidation of VP-16 to redox active species leads to enhanced genotoxic events linked to the known leukemogenic action of this anticancer agent. These MPO dependent effects are also dependent on GSH likely through thiyl radical formation and cycling. The protective effects of DHA and trolox further suggest that reduction of MPO-catalyzed VP-16 free radicals may be an effective strategy to prevent drug-induced second malignancies. Support: NIH R01 CA090787. Citation Format: Jason Goodspeed, Soumendra Karmahapatra, Ragu Kanagasabai, Alex Klausing, Jack C. Yalowich. Impact of antioxidants on myeloperoxidase (MPO)-dependent DNA damage and genotoxicity induced by etoposide (VP-16):implications for therapy-induced second malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1660. doi:10.1158/1538-7445.AM2015-1660