Abstract
The excessive release of heme during hemolysis contributes to the severity of sickle cell anemia (SCA) by exacerbating hemoglobin S (HbS) autoxidation, inflammation and systemic tissue damage. The present study investigated the effect of hydroxyurea (HU) on free radical neutralization and its stimulation of antioxidant genes in human peripheral blood mononuclear cells (PBMC) and human umbilical vein endothelial cells (HUVEC) in the presence or absence of hemin. HU (100 and 200 μM) significantly reduced the production of intracellular reactive oxygen species (ROS) induced by hemin at 70 μM in HUVEC. HUVECs treated with HU+hemin presented significant increases in nitric oxide (NO) production in culture supernatants. HU alone or in combination with hemin promoted the induction of superoxide dismutase-1 (SOD1) and glutathione disulfide-reductase (GSR) in HUVECs and PBMCs, and glutathione peroxidase (GPX1) in PBMCs. Microarray analysis performed in HUVECs indicated that HU induces increased expression of genes involved in the antioxidant response system: SOD2, GSR, microsomal glutathione S-transferase (MGST1), glutathione S-transferase mu 2 (GSTM2), carbonyl reductase 1 (CBR1) and klotho B (KLB). Significant increases in expression were observed in genes with kinase activity: protein kinase C beta (PRKCB), zeta (PRKCZ) and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta (PIK3C2B). HU also induced a significant increase in expression of the gene p62/sequestosome (p62/SQSTM1) and a significant decrease in the expression of the transcriptional factor BACH1 in HUVECs. Upstream analysis predicted the activation of Jun, miR-155-5p and mir-141-3p. These results suggest that HU directly scavenges free radicals and induces the expression of antioxidant genes via induction of the Nrf2 signaling pathway.
Highlights
Hydroxyurea (HU) is a hydroxylated analog of urea, which was initially identified as a myelosuppressive drug that acts by inhibiting ribonucleotide reductase
We investigated the effects of HU in human peripheral blood mononuclear cells (PBMC) and umbilical cord vein endothelial cells (HUVEC) pre-treated or not with hemin, an important pro-oxidant molecule released during hemolysis [3, 18, 19]
DPPH scavenging activity assays revealed that, despite higher IC50 values determined for HU at 100 and 200 μM, HU demonstrated considerable scavenging activity compared to controls
Summary
Hydroxyurea (HU) is a hydroxylated analog of urea, which was initially identified as a myelosuppressive drug that acts by inhibiting ribonucleotide reductase. After determining its antisickling effect, HU was approved in 1998 by the U.S Food and Drug Administration (FDA) for the treatment of sickle cell anemia (SCA). The imbalance caused by increased oxidation-reduction (redox) reactions in the vascular microenvironment in SCA provokes important deleterious effects [4]. Patients with SCA can present (i) intravascular and extravascular hemolysis with free heme release; (ii) autoxidation of HbS [3, 7]; (iii) nitric oxide (NO) depletion and endothelial dysfunction [8, 9]; (iv) ischemia-reperfusion events [10]; (v) marked leukocyte dysfunction, conferring a non-effector response against pathogens, and the dysregulation of inflammatory equilibrium that increases susceptibility to secondary infections [11,12,13]
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