Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells

Hydroxyurea (hydroxycarbamide, HU) arrests cells in the S-phase by inhibiting ribonucleotide reductase and DNA synthesis, significantly contributing to the release of nitric oxide (NO). We investigated the involvement of inducible NO synthase (NOS2) in the cytostatic effect of HU using in vitro shRNA-induced knockdown of the NOS2 transcript (NOS2_kd) or a specific NOS2 inhibitor (1400W) in human erythroleukemic HEL92.1.7 cells, as well as murine erythroid progenitors (mERPs) from HU-treated wild-type (WT) and Nos2 knockout (Nos2^-/-) mice. Over the long-term, HU increased NOS2 expression in HEL92.1.7 cells (via nuclear factor kappa B [NFκB] signaling) and in mERP. In the short-term, HU increased the activity of human recombinant and erythroleukemic cell-derived NOS2, as confirmed by NO metabolite nitrite/citrulline production. In silico molecular docking predicted that HU binds to the NOS2 active site and substrate L-arginine via hydrogen bonds. Molecular dynamics simulations showed reduced rigidity of the NOS2 active site upon interaction with HU, indicating stabilization of the enzyme-substrate complex. Both 1400W and NOS2_kd prevented the in vitro reduction in proliferation and induction of apoptosis in HEL92.1.7 cells by HU. NOS2_kd preferentially blocked early apoptosis and HU-induced S-phase arrest in HEL92.1.7 cells. The HU-induced decrease in proliferation and stimulation of early apoptosis in mERP were prevented in Nos2^-/- mice and by 1400W in WT mice. This study demonstrated that HU induces NOS2 activity through direct interaction and increased protein expression via NFκB signaling. Moreover, NOS2 mediates the HU-induced inhibition of proliferation and stimulation of apoptosis in erythroid cells.