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 Górska-Ponikowska , Agata Ploska , Dagmara Jacewicz , Michal Szkatula , Giampaolo Barone , Giosuè Lo Bosco , Fabrizio Lo Celso , Aleksandra Dąbrowska , Alicja Kuban-Jankowska , Monika Gorzynik-Debicka , Narcyz Knap , Lech Chmurzyński , Lawrence Wawrzyniec Dobrucki , Leszek Kalinowski , Michał Woźniak
Abstract2-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.
|Journal series||Redox Biology, ISSN 2213-2317, (N/A 140 pkt)|
|Publication size in sheets||0.50|
|License||Journal (articles only); published final; ; with publication|
|Score||= 140.0, 27-07-2020, ArticleFromJournal|
|Publication indicators||= 0.000; : 2017 = 1.863; : 2018 = 7.793 (2) - 2018=9.038 (5)|
|Citation count*||1 (2020-07-07)|
* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.