1. Modification of DNA structure by reactive nitrogen species as a result of 2-methoxyestradiol–induced neuronal nitric oxide synthase uncoupling in metastatic osteosarcoma cells
- Author
-
Lech Chmurzyński, Giampaolo Barone, Agata Płoska, Aleksandra Dabrowska, Michal Szkatula, Alicja Kuban-Jankowska, Dagmara Jacewicz, Fabrizio Lo Celso, Narcyz Knap, Michal Wozniak, Magdalena Gorska-Ponikowska, Lawrence W. Dobrucki, Giosuè Lo Bosco, Monika Gorzynik-Debicka, Leszek Kalinowski, Gorska-Ponikowska, Magdalena, Płoska, Agata, Jacewicz, Dagmara, Szkatula, Michal, Barone, Giampaolo, Lo Bosco, Giosue, Lo Celso, Fabrizio, Dabrowska, Aleksandra, Kuban-Jankowska, Alicja, Gorzynik-Debicka, Monika, Knap, Narcyz, Chmurzynski, Lech, Dobrucki, Lawrence Wawrzyniec, Kalinowski, Leszek, and Wozniak, Michal
- Subjects
0301 basic medicine ,DNA damage ,Clinical Biochemistry ,Bone Neoplasms ,Nitric Oxide Synthase Type I ,Nitric Oxide ,Biochemistry ,Nitric oxide ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Peroxynitrous Acid ,Humans ,MTT assay ,Viability assay ,lcsh:QH301-705.5 ,Reactive nitrogen species ,Settore CHIM/02 - Chimica Fisica ,Osteosarcoma ,lcsh:R5-920 ,Settore BIO/16 - Anatomia Umana ,Organic Chemistry ,DNA ,Reactive Nitrogen Species ,2-Methoxyestradiol ,Peroxynitrous acid ,030104 developmental biology ,chemistry ,lcsh:Biology (General) ,Settore CHIM/03 - Chimica Generale E Inorganica ,Cancer cell ,Biophysics ,lcsh:Medicine (General) ,030217 neurology & neurosurgery ,Peroxynitrite ,2 methoxyestradiol, nitric oxide, chemotherapy ,Research Paper - 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.
- Published
- 2020