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Modification of DNA structure by reactive nitrogen species as a result of 2-methoxyestradiol–induced neuronal nitric oxide synthase uncoupling in metastatic osteosarcoma cells
- Source :
- Redox Biology, Vol 32, Iss, Pp-(2020), Redox Biology
- Publication Year :
- 2020
- Publisher :
- Elsevier, 2020.
-
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.
- 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
Subjects
Details
- Language :
- English
- ISSN :
- 22132317
- Volume :
- 32
- Database :
- OpenAIRE
- Journal :
- Redox Biology
- Accession number :
- edsair.doi.dedup.....0452055dfbf00ffda95246a17f21cdbf