Abstract

Iron overload has been shown to increase mitochondrial dysfunction and apoptosis and may be implicated in leukocyte apoptosis. We assessed whether markers of leukocyte apoptosis and mitochondrial dysfunction are higher in iron-overloaded thalassemia patients compared with control subjects and whether improvement in the control of iron overload with deferasirox or deferoxamine (DFO) is associated with a change in the level of apoptotic markers.

Methods: Thalassemia Clinical Research Network patients participating in the Novartis CICL670A0107 trial (a randomized comparison of deferasirox, an oral iron chelator, vs. DFO) were eligible and 44 (25 male, 21.8 yrs) were enrolled in the study. Fasting blood samples were obtained

  • after a 5-day washout of DFO prior to commencing treatment with study drug, and

  • 24 hours post-chelator at 1, 6, and 12 months on study.

Thirty healthy controls matched for age, sex, race and antioxidant usage (15 male, 24.5 yrs) also supplied a blood sample at baseline. After blood collection, plasma, serum and cells were separated by centrifugation. The pro-apoptotic marker Bax (an inducer of mitochondrial dysfunction) and the anti-apoptotic marker Bcl-2 were determined by ELISA. A high ratio of Bax/Bcl-2 indicates decreased stability of the mitochondrial outer membrane and increased potential for mitochondrial dysfunction and apoptosis. Activity of caspase–3 and −9 (both pro-apoptotic) were determined by a luminescent enzyme activity assay and reported as the average relative light units (RLU)/μg protein. Apoptotic markers were log-transformed prior to analysis and means and % change are reported.

Results: At baseline, thalassemia patients had elevated Bax compared to the controls (17.4 vs. 11.6 pg/μg protein, p = 0.006). Similarly the activity of caspase-3 and −9 was high relative to the controls (for caspase-3, 1823 vs. 1041 RLU/μg protein, p = 0.01; and for caspase-9, 2482 vs. 1322 RLU/μg protein, p = 0.001). In longitudinal analysis, liver iron concentration and ferritin declined in both treatment groups (p ≤ 0.02 for both). This paralleled a decline in the ratio of Bax/Bcl-2 (–27.3% /yr, average decline, p = 0.033) and ALT (–7.3% /yr, average decline, p = 0.040). There was no significant difference between chelators in the rate of change of these markers over time.

Conclusions: These findings demonstrate that markers of leukocyte apoptosis and mitochondrial dysfunction are high in thalassemia compared to controls and that the Bax/Bcl-2 ratio (a marker of mitochondrial dysfunction in apoptosis) decreased with effective iron chelation. DFO and deferasirox showed equal effectiveness in decreasing iron burden, ALT and the ratio of Bax/Bcl-2. Thus, with effective chelation, it may be possible to improve leukocyte and mitochondrial function and the levels of apoptosis.

Author notes

Disclosure:Employment: Daniele Alberti and Jaymes Holland are employed by Novartis. Research Funding: Paul Harmatz and Elliott Vichinsky have research funding from Novartis.