Deferasirox is a powerful NF-kappaB inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging

Abstract

Background: Usefulness of iron chelation therapy in myelodysplastic patients is still under debate but many authors suggest its possible role in improving survival of low-risk myelodysplastic patients. Several reports have described an unexpected effect of iron chelators, such as an improvement in hemoglobin levels, in patients affected by myelodysplastic syndromes. Furthermore, the novel chelator deferasirox induces a similar improvement more rapidly. Nuclear factor-kappaB is a key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including myelodysplastic syndromes.

Design and methods: We evaluated deferasirox activity on nuclear factor-kappaB in myelodysplastic syndromes as a possible mechanism involved in hemoglobin improvement during in vivo treatment. Forty peripheral blood samples collected from myelodysplastic syndrome patients were incubated with 50 muM deferasirox for 18h.

Results: Nuclear factor-kappaB activity dramatically decreased in samples showing high basal activity as well as in cell lines, whereas no similar behavior was observed with other iron chelators despite a similar reduction in reactive oxygen species levels. Additionally, ferric hydroxyquinoline incubation did not decrease deferasirox activity in K562 cells suggesting the mechanism of action of the drug is independent from cell iron deprivation by chelation. Finally, incubation with both etoposide and deferasirox induced an increase in K562 apoptotic rate.

Conclusions: Nuclear factor-kappaB inhibition by deferasirox is not seen from other chelators and is iron and reactive oxygen species scavenging independent. This could explain the hemoglobin improvement after in vivo treatment, such that our hypothesis needs to be validated in further prospective studies.

Figure 1.

Deferasirox inhibits NF-kB activity in K562 cells whereas deferioxamine and deferiprone do not. (A) Immunofluorescence assay using p65 antibody in control cells (a) and after incubation with deferasirox 50 mM for 18 h (b–c). Nuclei are stained in red, the green signal represents p65 subunit. In control cells the NF-κB subunit is mainly localized in the nucleus as indicated by the yellow signal whereas, after incubation with the drug, p65 subunit is localized in the cytoplasm in the inactive form as indicated by the green signal in the cytoplasm and the absence of yellow signal in the nucleus. The graph represents the signal intensity quantification in both the cellular compartments. Nuclear signal intensity is significantly different in control and incubated cells (P<0.001). (B) Western blotting using p65 antibody for the detection of proteins in either nuclear (N) or cytoplasmic (C) extracts in K562 cells. The upper line indicates the p65 antibody, while both the lower lines represent the internal controls for either cytoplasmic (actin antibody) or nuclear extracts (lamin antibody). p65 nuclear localization is decreased only after deferasirox incubation but neither deferioxamine nor deferiprone incubation. The graph shows the quantification of the signal intensity detected by Western blotting. (C) Immunofluorescence assay using p65 antibody in K562 cells before and after incubation with deferioxamine and deferiprone either at 0.5 mM for 30 min or 50 mM for 18 h. After all the different conditions of incubation, p65 remains localized in the nucleus in the active form as in control cells. The graph illustrates NF-κB signal intensity in all the different conditions of incubation. No statistically significant differences have been found between control values and K562 treated cells with both the drugs. (D) Western blotting using p65 antibody for the detection of proteins in either cytoplasmic or nuclear extracts in K562 cells. The upper line indicates the p65 antibody, while the lower line represents an internal control for either cytoplasmic (actin antibody) or nuclear extracts (lamin antibody). p65 nuclear localization is not decreased after deferioxamine incubation for 30 min.

Figure 2.

(A) NF-κB DNA binding activity (EMSA assay) in K562 cells treated either by deferasirox 50 mM for 18 h or by deferioxamine and deferiprone both at 0.5 mM for 30 min. Only after deferasirox incubation is the DNA binding activity of p65 subunit decreased. The first two lanes represent K562 cells incubated with the NF-κB inhibitor PS 1145 as negative control. The black arrow indicates specific binding to the consensus sequence. The graph on the right represents densitometry data. (B) EMSA assay for NF-κB DNA binding activity in K562 cells in basal conditions and after incubation with deferoxamine and deferiprone 50 mM both for 18 h. The DNA binding activity (indicated by the black arrow) is not decreased in both the different conditions of incubation with respect to the one detected in control cells. The graph on the right represents densitometry data.

Figure 3.

Deferasirox inhibits NF-κB activity in MDS patients. (A) NF-kB DNA binding activity (EMSA assay) in a MDS patient: NF-κB activity can be detected in control cells and its amount decreases after 18 h of deferasirox incubation in vitro at 50 mM. The black arrow indicates specific binding to the consensus sequence. The second lane of each condition was loaded with an unspecific cold probe as negative control. (B) Inhibition of NF-κB activity after in vitro incubation with 50 mM deferasirox of mononuclear PB cells of MDS patients for 18 h, as measured by ELISA assay. In all patients with high basal NF-κB activity (28 patients out of 40 tested), it is possible to detect transcription factor (TF) inhibition after drug incubation. (C) Immunofluorescence assay of PB cells from an MDS patient. At basal conditions on the left, p65 is completely localized in the nucleus, but after deferasirox incubation, the subunit is largely located in the cytoplasm, as demonstrated by the presence of the green signal.

Figure 4.

Deferasirox inhibits NF-κB activity independently from cell iron deprivation by chelation and reactive oxygen species scavenging. (A) ROS assay by DCF staining in K562 cells before and after incubation with three chelators at the conditions previously described. Mean fluorescence intensities (MFI) of K562 untreated cells and after incubation with deferioxamine, deferiprone and deferasirox are shown in the two graphs. There is a statistically significant difference comparing MFI samples before and after incubation with the three different drugs. Conversely, no statistically significant difference can be detected comparing MFI after deferasirox incubation with respect to either deferioxamine or deferiprone incubation. (B) The graph shows NF-κB activity measured by ELISA assay in K562 cells incubated respectively with deferasirox 50 mM alone, ferric hydroxyquinoline (FHQ) at 2.5 mM and 5 mM and with both deferasirox and FHQ. No statistically significant difference in NF-κB activity reduction has been detected in samples incubated by both deferasirox and FHQ with respect to those treated by deferasirox alone, suggesting a mechanism of action of the drug whch is independent from cell iron deprivation by chelation.

Figure 5.

Apoptosis evaluated by fluorescence-activated cell sorting for the detection of annexin V-positive K562 cells after incubation with the three chelators and with etoposide and deferasirox. The apoptotic rate, represented by the percentage of cells both positive for Annexin V and negative for propidium iodide by FACS is indicated in each graph and represented on the right. (A) The apoptotic rate is not increased with respect to basal conditions after incubation with deferioxamine and deferiprone 0.5 mM for 30 min and deferasirox 50 mM for 18 h. The same results have been obtained after deferioxamine 50 mM incubation and deferiprone 50 and 100 mM incubation for 18 h. (B) Apoptosis assay of K562 cells in control cells and after etoposide 10 mM for 72 h alone or preceded by 50 mM deferasirox incubation for 18 h. After etoposide 10 mM for 72 h incubation an increase in the apoptotic cells amount can be detected (mean value 4.68%±0.46) and this is even higher if etoposide is preceded by deferasirox 50 mM for 18 h incubation (mean value 9.13±0.2). K562 cells were also incubated with etoposide preceeded by deferioxamine and deferiprone 50 mM for 18 h and 0,5 mM for 30 min without any statistically significant increase in the apoptotic rate as shown also by the graph on the right. No changes in apoaptotic rate can be detected after deferioxamine incubation (mean 5.1±0.64 at 50 mM and 4.3±0.49 at 50 mM), whereas deferiprone induces a slight increase in apoptotic rate (mean 6.7±0.45 at 0.5 mM and 6.8±0.7 at 50 mM).