3222 – MEDIATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN INHIBITION OF PROLIFERATION AND STIMULATION OF APOPTOSIS BY HYDROXYUREA IN ERYTHROID CELLS

• Published in: Experimental hematology 2024-08, Vol.137, p.104542, Article 104542
• Main Authors: Čokić, Vladan, Dragojević, Teodora, Živković, Emilija, Milenković, Dejan, Vukotić, Milica
• Format: Article
• Language: English
• ISSN: 0301-472X
• DOI: 10.1016/j.exphem.2024.104542

Hydroxyurea (HU) is a cytostatic used in the treatment of sickle cell anemia and thalassemia according to its stimulation of fetal hemoglobin. HU acts as a nitric oxide (NO) donor after oxidation in the presence of hem proteins, and moreover induces the expression and activity of endothelial NO synthase (NOS). We investigated the dependence of inducible NOS (NOS2) on HU regulation of proliferation and apoptosis in vitro in human erythroleukemic cells HEL92.1.7 and ex vivo in murine erythroid progenitors (mERP) from Nos2 knockout mice. Molecular docking simulation was used to evaluate the interactions between amino acids in the active binding sites of NOS and HU. Using shRNA-transformed knock-down of NOS2, we prevented HU induced inhibition of proliferation and S-phase arrest of HEL92.1.7 cells. NOS2 shRNA and Nos2 knockout preferentially blocked early apoptosis of HEL92.1.7 cells and mERP, respectively. The Nos2 knockout prevented HU induced decrease of proliferation and induction of apoptosis in mERP. HU activated nuclear factor kappa B (Nf-κB) and p38 mitogen-activated protein kinase (MAPK) signalling in HEL92.1.7 cells, while HU stimulation of NOS2 expression was Nf-κB dependent. Molecular dynamic simulation showed a slight decrease in the NOS2 receptor rigidity upon HU binding, confirming the stability of HU at the active site of the NOS2 receptor through amino acids ASP382, ASP385 and ARG388. This study demonstrated NOS2 dependence in HU inhibition of proliferation and stimulation of apoptosis of erythroid cells.