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
Affiliations
Magdalena Gorska-Ponikowska
Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211, Gdansk, Poland; Euro-Mediterranean Institute of Science and Technology, Palermo, Italy; Department of Biophysics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, Germany; Corresponding author. Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211, Gdansk, Poland.
Agata Ploska
Department of Medical Laboratory Diagnostics, Medical University of Gdansk, Gdansk, Poland; Biobanking and Biomolecular Resources Research Infrastructure Poland (BBMRI.PL), Gdansk, Poland
Dagmara Jacewicz
Department of General and Inorganic Chemistry, University of Gdansk, Gdansk, Poland
Michal Szkatula
Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211, Gdansk, Poland
Giampaolo Barone
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo,Palermo, Italy
Giosuè Lo Bosco
Euro-Mediterranean Institute of Science and Technology, Palermo, Italy; Department of Mathematics and Computer Science, University of Palermo, Palermo, Italy
Fabrizio Lo Celso
Department of Physics and Chemistry ''Emilio Segrè'', University of Palermo, Palermo, Italy
Aleksandra M Dabrowska
Department of Bioinorganic Chemistry University of Gdansk, Gdansk, Poland
Alicja Kuban-Jankowska
Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211, Gdansk, Poland
Monika Gorzynik-Debicka
Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211, Gdansk, Poland
Narcyz Knap
Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211, Gdansk, Poland
Lech Chmurzynski
Department of General and Inorganic Chemistry, University of Gdansk, Gdansk, Poland
Lawrence Wawrzyniec Dobrucki
Department of Medical Laboratory Diagnostics, Medical University of Gdansk, Gdansk, Poland; Biobanking and Biomolecular Resources Research Infrastructure Poland (BBMRI.PL), Gdansk, Poland; Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Beckman Institute for Advanced Science and Technology, Urbana, IL, USA
Leszek Kalinowski
Department of Medical Laboratory Diagnostics, Medical University of Gdansk, Gdansk, Poland; Biobanking and Biomolecular Resources Research Infrastructure Poland (BBMRI.PL), Gdansk, Poland
Michal Wozniak
Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211, Gdansk, Poland
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.
