Functional Analysis of the FOXO3 Gene on the Induction of Fetal Hemoglobin in K562 Cells

The β-hemoglobinopathies are known as genetic disorders of high impact and wide distribution worldwide, with chronic and variable clinical prognosis. These pathologies have their symptoms and sequelae alleviated by increased levels of fetal hemoglobin (HbF; α2γ2). The Hydroxyurea, a drug used for the treatment of β-hemoglobinopathies, is effective for the maintenance and control of the development of the main clinical manifestations, however, it needs some prerequisites for its use, which indicates the need for new therapeutic approaches for this group of individuals. The family of Forkhead Box O transcription factors (FOXO), composed of FOXO1, FOXO3, FOXO4 and FOXO6, plays a key role in the regulation of several biological processes such as: regulation of the cell cycle, modulation oxidative stress, regulation mechanisms of response to DNA damage, controlling apoptosis and inflammatory response. Among members of the FOXO family, FOXO3 has been described as a physiological regulator of the erythroid maturation process and indicated as a positive regulator of HbF levels. In the present work, we evaluated the expression levels of the FOXO3 gene in patients with hemoglobinopathies and, in an attempt to better understand the pathophysiology of the disease, we performed a subcloning of the FOXO3 gene into a lentiviral expression system for its constitutive expression in vitro and subsequent evaluation of its activity as a possible transcription factor for γ globin genes (HBG1 and HBG2). In our clinical study, FOXO3 expression was significantly higher in patients with sickle cell anemia when compared to healthy donors (HbAA) (P <0.001) . In addition, a differential expression of FOXO3 was associated with the development of lower limb ulcer (P <0.005). Although we observed a tendency to increase FOXO3 expression in patients with 2 or more clinical manifestations, this association was not statistically significant (P = 0.06). The same was observed when we compared the number of clinical complications presented by patients last year with HbF levels: patients with 2 or more complications had lower levels of HbF, although this difference did not reach statistical significance (P = 0.182). There was no correlation between FOXO3 expression levels and HbF dosage (r = -0.12). Because of the activity of the FOXO3 gene in the control of oxidative stress, experiments were performed to evaluate the ROS and NO rates in the cell culture and a significant role was found, the FOXO3 increasing NO production (P = 0.0001) as well as in control of ROS produced in mitochondria.