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

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.