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.

2-甲氧基雌二醇（2-ME）是一种生理抗癌化合物，是17β-雌二醇的代谢产物。以前，我们的研究小组证明，从机理的角度来看，2-ME的抗癌作用机制之一是神经元一氧化氮合酶（nNOS）的特异性诱导和核劫持，导致局部产生硝基氧化应激，最后导致癌细胞死亡。

目前的研究旨在建立2-ME产生活性氮物质的基本机理。我们进一步实现了鉴定2-ME DNA破坏机制中涉及的特定反应性氮物种。

该研究使用转移性骨肉瘤143B细胞进行。我们检测生物活性的（自由）的一氧化氮的释放（^• NO）与过氧亚硝酸盐的并发测量（ONOO ^- ）实时地通过使用在143B细胞系的单细胞^• NO / ONOO ^-敏感的微传感器用钙离子载体刺激后。二氧化氮的检测（^• NO ₂）和化学速率常数的测定通过停止流技术进行。通过密度泛函理论计算来评估反应性氮物种对DNA鸟嘌呤碱基的亲和力。使用成像细胞术确定核因子NF-kB的表达和定位，同时通过MTT测定评估细胞活力测定。

在本文中，我们提出了2-ME通过增加细胞反应性氮的过量生产来触发促凋亡信号级联反应-nNOS蛋白水平增加的酶解偶联作用的结果。特别是，我们证明，ONOO ^-和^• NO ₂从过氧亚硝酸（ONOOH）和/或通过自动氧化直接形成^• NO，在的2-ME抗癌机理DNA损伤的诱导物。具体地，朝向DNA的鸟嘌呤碱基，通过密度泛函理论计算评价反应性氮物种的亲和力，在为了降低：ONOOH> ONOO ^-  >  ^• NO ₂  >  ^• NO。

因此，我们建议考虑将nNOS的特异性诱导剂作为化学疗法领域的有效工具。