Effect Of Deferasirox On Reactive Species Of Oxygen (ROS) Production In Hematopoietic Stem Cells: Up Or Down?

Deferasirox (DFX) is an iron chelator used to prevent and treat complications related to transfusional iron overload in myelodisplastic syndrome patients (MDS). Intriguingly, DFX treatment induces haematological responses in a consistent percentage of patients whereas other chelators, like deferoxamine (DFO) do not show such an appreciable effect. A body of literature documents a general reduction of oxidative stress in patients treated with DFX but little is known about the direct effect of DFX treatment on hematopoietic stem cells (HSC). Consolidated evidence highlights the importance of redox signalling in the homeostasis of fundamental processes, particularly in controlling the balance between self-renewal and differentiation of stem cells. In this setting, reactive species of oxygen (ROS) would act as secondary messengers, modulating the expression of master transcription factors and regulatory proteins leading or (pre)conditioning stem cells towards differentiation. In the present study we investigated the effect of DFX and DFO on ROS production in hematopoietic stem/progenitor cells (HSPCs) in order to identify a molecular mechanism explaining the differential effect of iron chelators in rescuing altered hematopoiesis.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy subjects and incubated for 24 hours with DFO and DFX in a range of doses between 6.2mM and 200mM. Human HSPCs, isolated upon informed consent from peripheral blood of G-CSF-treated healthy donors by immuno-selection against the specific markers CD133 and CD34, were treated with 100 mM DFX or DFO for 24 hours.

To completely abrogate ROS production, cells were co-incubated with diphenil iodide (DPI) 100mM, a known inhibitor of the main proteins able to generate ROS: the flavo-oxidases of the respiratory chain and the NADPH oxidases. Cell viability was determined by trypan blue staining. ROS levels were analyzed by laser scanning confocal microscopy (LSCM) and flow cytometry after the incubation at 37°C for 15 minutes with the intracellular H₂O₂ specific probe dichlorodihhyrofluorescein-diacetate (H₂DCFDA) 10mM. b-catenin and BMI1 protein levels were assessed by western blotting. Data were presented as mean±s.e.m. and were compared by unpaired Student T-Test; a p<0.05 was considered significant.

The flow cytometry analysis on PBMCs revealed that DFX, surprisingly, was able to induce ROS production in a dose-dependent manner with a significant increase at 100mM and 200mM compared to CTRL (p=0.02) whereas DFO treatment didn’t show any effect compared to CTRL. Since the cell viability resulted to be not affected by both drugs, the dose of 100mM was chosen for the following experiments performed with HSPCs where the ability of DFX to cause a significant increase of H2O2 was confirmed. A deeper analysis aimed to clarify the localization of ROS released following DFX treatment was performed by LSCM: a significant up-regulation of mitochondrial ROS induced by DFX and not by DFO was clearly observed compared to untreated samples. Importantly, the addition of DPI strongly reduced the entity of the signal detected by DCF (p=0.01 versus DFX).

In order to understand a possible link between ROS production and the ability of DFX to restore the hematopoiesis, the expression of b-catenin and BMI1, both of them sensitive to the intracellular redox state and involved in the hematopoietic function, was analysed by western blotting. It was shown that DFX significantly reduced the expression of both b-catenin, strictly linked to hematopoietic function and BMI1 (p=0.02 versus CTRL), a regulatory protein sustaining immaturity and required for the maintaining of adult self-renewing hematopoietic stem cells. Conversely, no significant variation was observed by DFO treatment. Interestingly, the b-catenin and BMI-1expression downregulations, specifically induced by DFX, were completely reversed by ROS abrogation obtained by DPI treatment.

Our results show, for the first time, that DFX treatment, independently on its iron-chelating property, is able to induce ROS production that in turn influences key factors involved in self-renewal/differentiation of hematopoietic stem cells. In this scenario, the modulation of ROS, because of their ability to restore the hematopoietic function, could be taken in account as potential further pharmacological target in MDS treatment.