Modification of DNA structure by reactive nitrogen species as a result of 2-methoxyestradiol–induced neuronal nitric oxide synthase uncoupling in metastatic osteosarcoma cells

Magdalena Gorska-Ponikowska,Agata Ploska,Dagmara Jacewicz,Michal Szkatula,Giampaolo Barone,Giosuè Lo Bosco,Fabrizio Lo Celso,Aleksandra M Dabrowska,Alicja Kuban-Jankowska,Monika Gorzynik-Debicka,Narcyz Knap,Lech Chmurzynski,Lawrence Wawrzyniec Dobrucki,Leszek Kalinowski,Michal Wozniak

doi:10.1016/j.redox.2020.101522

Abstract

2-methoxyestradiol (2-ME) is a physiological anticancer compound, metabolite of 17β-estradiol. Previously, our group evidenced that from mechanistic point of view one of anticancer mechanisms of action of 2-ME is specific induction and nuclear hijacking of neuronal nitric oxide synthase (nNOS), resulting in local generation of nitro-oxidative stress and finally, cancer cell death.The current study aims to establish the substantial mechanism of generation of reactive nitrogen species by 2-ME. We further achieved to identify the specific reactive nitrogen species involved in DNA-damaging mechanism of 2-ME.The study was performed using metastatic osteosarcoma 143B cells. We detected the release of biologically active (free) nitric oxide (•NO) with concurrent measurements of peroxynitrite (ONOO−) in real time in a single cell of 143B cell line by using •NO/ONOO− sensitive microsensors after stimulation with calcium ionophore. Detection of nitrogen dioxide (•NO2) and determination of chemical rate constants were carried out by a stopped-flow technique. The affinity of reactive nitrogen species toward the guanine base of DNA was evaluated by density functional theory calculations. Expression and localization of nuclear factor NF-kB was determined using imaging cytometry, while cell viability assay was evaluated by MTT assay.Herein, we presented that 2-ME triggers pro-apoptotic signalling cascade by increasing cellular reactive nitrogen species overproduction – a result of enzymatic uncoupling of increased nNOS protein levels. In particular, we proved that ONOO− and •NO2 directly formed from peroxynitrous acid (ONOOH) and/or by auto-oxidation of •NO, are inducers of DNA damage in anticancer mechanism of 2-ME. Specifically, the affinity of reactive nitrogen species toward the guanine base of DNA, evaluated by density functional theory calculations, decreased in the order: ONOOH > ONOO− > •NO2 > •NO.Therefore, we propose to consider the specific inducers of nNOS as an effective tool in the field of chemotherapy.

Highlights

The current study aims to establish the substantial mechanism of generation of reactive nitrogen species by 2-ME
We proved that ONOO− and NO2 directly formed from peroxynitrous acid (ONOOH) and/or by autooxidation of NO, are inducers of DNA damage in anticancer mechanism of 2-ME
We further evaluated that 2-ME-cancer cell death is strictly associated with DNA damage and genomic instability due to nuclear localization of neuronal nitric oxide synthase (nNOS) [12]

Summary

Introduction

The current study aims to establish the substantial mechanism of generation of reactive nitrogen species by 2-ME. We further achieved to identify the specific reactive nitrogen species involved in DNA-damaging mechanism of 2-ME. We presented that 2-ME triggers pro-apoptotic signalling cascade by increasing cellular reactive nitrogen species overproduction – a result of enzymatic uncoupling of increased nNOS protein levels. We proved that ONOO− and NO2 directly formed from peroxynitrous acid (ONOOH) and/or by autooxidation of NO, are inducers of DNA damage in anticancer mechanism of 2-ME. The affinity of reactive nitrogen species toward the guanine base of DNA, evaluated by density functional theory calculations, decreased in the order: ONOOH > ONOO− > NO2 > NO. We propose to consider the specific inducers of nNOS as an effective tool in the field of chemotherapy. Serum levels of 2-ME range from 30 pM in men to as much as over 30 nM in pregnant women [1,2,3]; while, pharmacologically active plasma concentrations are equaled to μM concentrations [1,2,3]

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