Fetal hemoglobin induction through the suppressor of lsd1 by using gsk-lsd1 that now is epigenetic drugs
,1,* Mojtaba khayam nekouie
,2 Mohammad hamid
,3 Majid sadeghizadeh
1. ٍResearcher in Department of Medical Genetic in MOM Center
2. Faculty of Biological Science, Tarbiat Modares University
3. iotechnology Research Center, Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran
4. Faculty of Biological Science, Tarbiat Modares University
Introduction: hemoglobinopathies, b-thalassemia and sickle cell disease (scd) together include the most common inherited diseases. the only current therapy for scd is treatment with hydroxyurea (hu). hu induces fetal hemoglobin (hbf) synthesis in about half of treated patients by an as yet unknown mechanism(s), and its long-term effects are largely unknown and in some patients have the bad effect. switch from fetal γ globin to β globin gene expression occurs at birth, hbf induction is known clinically to reduce organ morbidity and pain in scd patients, and to inhibit sickle polymer formation and consequently the destruction of erythrocytes in vitro. several adult-stage γ globin repressors, such as bcl11a, ikaros, gata1 and sox6 have been identified that interact with each other to repress the γ globin genes. for the past decade, several adult-stage γ globin repressors, such as bcl11a, ikaros, gata1 and sox6 that called dred have been identified that interact with each other to repress the γ globin genes. the strong recent results showed one of the modifying enzymes is lsd1, a monoamine oxidase that removes activating chromatin signatures, thereby leading to gene repression. recently result showed that a highly specific inhibitor for lsd1 called tranylcypromine (tc). in human cd34+ hematopoietic stem cells induced to differentiate into erythroid cells in vitro, hbf was induced to therapeutic levels in a tc dosage-dependent manner. filed a novel use patent for tc, which is already fda approved and off patent, and intend to test for cryptic properties (hbf induction) in cells and sickle cell mice.
Material method: we examined the effects of the gsk-lsd1 inhibitor on cd34+ cells are isolated from cord blood using positive immunomagnetic separation techniques, cells ex vivo. cell number and viability were determined by trypan blue staining. cell morphology was examined by wright-giemsa staining (sigma-aldrich). flow-cytometric analysis showed that hbf was induced in all of the cells in a dose-dependent manner.
we treated the cells with 0, 0.5, 1.5, and 5 μm of the gsk-lsd1 inhibitor on days 4 to 14 of the differentiation culture. then we performed an analysis of the expression of lsd1 and γ globin genes comparable levels throughout differentiation with real-time pcr using the iq sybr green master mix.
Results: after treatment gsk-lsd1 inhibitor at 0.5, 1.5, and 5 μm did not alter cell proliferation or viability, but 5μm gsk-lsd1 reduced cell proliferation and delayed differentiation without affecting cell viability. in 1.5-μm concentration of the gsk-lsd1 inhibitor, the mean of γ-globin mrna expression was induced up to 33-fold. we observed a decrease in the lsd1 mrna expression in a 5-μm concentration of the gsk-lsd1 inhibitor.
Conclusion: our results indicated that lsd1 played an important role in γ-globin silencing in adult erythroid cells. further, the gsk- lsd1 inhibitor increase concentration of hbf induction within the therapeutic plasma concentration. finally lsd1 is thus a promising therapeutic target for γ-globin induction, and gsk- lsd1 inhibitor lead compound for the development of a new γ-globin inducer. thus, with continued careful attention to fundamental biological and pharmacologic considerations, there is potential that rational, molecular- targeted, safe and highly potent disease-modifying therapy can be realized for patients with sickle cell disease, with the accessibility and cost-effective properties needed for worldwide effect.
Fetal hemoglobin, hemoglobinopathies, lsd1, gsk-lsd1, epigenetic drugs