Deferasirox shows in vitro and in vivo antileukemic effects on murine leukemic cell lines regardless of iron status

Numerous studies have shown the antiproliferative effect of iron chelating agents (ICAs), which have been used traditionally in patients with secondary iron overload (SIO). Because the in vivo model for these studies has been animals with normal iron status, the antileukemic effect of ICAs in the SIO condition has not been determined clearly. We investigated the in vitro and in vivo effects of ICAs in murine leukemic cell lines regarding the iron status. The viability of both EL4 cells and L1210 cells incubated with either deferoxamine (DFO) or deferasirox (DFX) decreased in a concentration-dependent manner. This effect was most prominent in L1210 cells treated with DFX. The viability of L1210 cells incubated with both ICAs did not change regardless of the presence of ferric chloride. The percentage of apoptosis in L1210 cells treated with DFO or DFX increased in a concentration-dependent manner; however, the expression of Fas showed no significant change. The non-SIO mice and SIO mice bearing L1210 cells showed longer survival than other groups when treated with DFX, whereas the SIO mice treated with DFO showed shorter survival than the control group. The tumor was significantly smaller in the SIO mice treated with DFX or DFO compared with the control group. The iron content of the liver or the tumor in SIO mice decreased after ICA treatment. This study indicates an antileukemic effect of DFX regardless of iron status and suggests that the use of DFX has a survival benefit for SIO leukemia murine model in terms of iron chelation and antileukemic therapy. Numerous studies have shown the antiproliferative effect of iron chelating agents (ICAs), which have been used traditionally in patients with secondary iron overload (SIO). Because the in vivo model for these studies has been animals with normal iron status, the antileukemic effect of ICAs in the SIO condition has not been determined clearly. We investigated the in vitro and in vivo effects of ICAs in murine leukemic cell lines regarding the iron status. The viability of both EL4 cells and L1210 cells incubated with either deferoxamine (DFO) or deferasirox (DFX) decreased in a concentration-dependent manner. This effect was most prominent in L1210 cells treated with DFX. The viability of L1210 cells incubated with both ICAs did not change regardless of the presence of ferric chloride. The percentage of apoptosis in L1210 cells treated with DFO or DFX increased in a concentration-dependent manner; however, the expression of Fas showed no significant change. The non-SIO mice and SIO mice bearing L1210 cells showed longer survival than other groups when treated with DFX, whereas the SIO mice treated with DFO showed shorter survival than the control group. The tumor was significantly smaller in the SIO mice treated with DFX or DFO compared with the control group. The iron content of the liver or the tumor in SIO mice decreased after ICA treatment. This study indicates an antileukemic effect of DFX regardless of iron status and suggests that the use of DFX has a survival benefit for SIO leukemia murine model in terms of iron chelation and antileukemic therapy. Iron is one of the essential elements for maintaining normal cellular function and is involved in various electron transfer systems. Patients receiving multiple red blood cell transfusions are at risk for developing secondary iron overload (SIO), because humans do not have a physiologic mechanism to excrete excess iron [1Fleming R.E. Ponka P. Iron overload in human disease.N Engl J Med. 2012; 366: 348-359Crossref PubMed Scopus (394) Google Scholar]. Because SIO has been associated with fatal diseases such as cardiomyopathy and hepatic fibrosis, the development of iron chelating agents (ICAs) has been focused on their use in SIO treatment [2Kushner J.P. Porter J.P. Olivieri N.F. Secondary iron overload.Hematology Am Soc Hematol Educ Program. 2001; : 47-61Crossref PubMed Scopus (206) Google Scholar, 3Kalinowski D.S. Richardson D.R. The evolution of iron chelators for the treatment of iron overload disease and cancer.Pharmacol Rev. 2005; 57: 547-583Crossref PubMed Scopus (591) Google Scholar]. Recently, the role of ICAs as antitumor agents was investigated by targeting ribonucleotide reductase (RR) [3Kalinowski D.S. Richardson D.R. The evolution of iron chelators for the treatment of iron overload disease and cancer.Pharmacol Rev. 2005; 57: 547-583Crossref PubMed Scopus (591) Google Scholar, 4Buss J.L. Torti F.M. Torti S.V. The role of iron chelation in cancer therapy.Curr Med Chem. 2003; 10: 1021-1034Crossref PubMed Scopus (192) Google Scholar], which is the iron-dependent enzyme involved in the rate limiting phase of DNA synthesis. As a result, rapidly proliferating cells, such as cancer cells, have a higher requirement for iron and are highly vulnerable to iron depletion by ICAs. Various ICAs have been investigated for their anticancer activity in cell culture experiments, animal models, and human clinical trials [5Blatt J. Stitely S. Antineuroblastoma activity of desferoxamine in human cell lines.Cancer Res. 1987; 47: 1749-1750PubMed Google Scholar, 6Becton D.L. Roberts B. Antileukemic effects of deferoxamine on human myeloid leukemia cell lines.Cancer Res. 1989; 49: 4809-4812PubMed Google Scholar, 7Blatt J. Deferoxamine in children with recurrent neuroblastoma.Anticancer Res. 1994; 14: 2109-2112PubMed Google Scholar, 8Whitnall M. Howard J. Ponka P. Richardson D.R. A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics.Proc Natl Acad Sci U S A. 2006; 103: 14901-14906Crossref PubMed Scopus (408) Google Scholar, 9Ba Q. Hao M. Huang H. et al.Iron deprivation suppresses hepatocellular carcinoma growth in experimental studies.Clin Cancer Res. 2011; 17: 7625-7633Crossref PubMed Scopus (49) Google Scholar]. Deferoxamine (DFO), a hexadentate siderophore, has been used widely for the treatment of SIO. Various studies have shown the in vitro and in vivo antiproliferative activity of DFO against a wide range of tumors, including hepatomas, leukemias, and neuroblastomas [5Blatt J. Stitely S. Antineuroblastoma activity of desferoxamine in human cell lines.Cancer Res. 1987; 47: 1749-1750PubMed Google Scholar, 6Becton D.L. Roberts B. Antileukemic effects of deferoxamine on human myeloid leukemia cell lines.Cancer Res. 1989; 49: 4809-4812PubMed Google Scholar, 7Blatt J. Deferoxamine in children with recurrent neuroblastoma.Anticancer Res. 1994; 14: 2109-2112PubMed Google Scholar, 9Ba Q. Hao M. Huang H. et al.Iron deprivation suppresses hepatocellular carcinoma growth in experimental studies.Clin Cancer Res. 2011; 17: 7625-7633Crossref PubMed Scopus (49) Google Scholar]. However, the high hydrophilicity and short half-life of DFO limits its duration and the choice for its route of administration [2Kushner J.P. Porter J.P. Olivieri N.F. Secondary iron overload.Hematology Am Soc Hematol Educ Program. 2001; : 47-61Crossref PubMed Scopus (206) Google Scholar, 3Kalinowski D.S. Richardson D.R. The evolution of iron chelators for the treatment of iron overload disease and cancer.Pharmacol Rev. 2005; 57: 547-583Crossref PubMed Scopus (591) Google Scholar, 4Buss J.L. Torti F.M. Torti S.V. The role of iron chelation in cancer therapy.Curr Med Chem. 2003; 10: 1021-1034Crossref PubMed Scopus (192) Google Scholar]. For this reason, deferasirox (DFX), a newly developed oral ICA, is of interest, because of its significant antiproliferative activity against hepatoma and myeloid malignancies [10Lescoat G. Chantrel-Groussard K. Pasdeloup N. Nick H. Brissot P. Gaboriau F. Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism.Cell Prolif. 2007; 40: 755-767Crossref PubMed Scopus (38) Google Scholar, 11Kim J.L. Kang H.N. Kang M.H. Yoo Y.A. Kim J.S. Choi C.W. The oral iron chelator deferasirox induces apoptosis in myeloid leukemia cells by targeting caspase.Acta Haematol. 2011; 126: 241-245Crossref PubMed Scopus (21) Google Scholar, 12Ohyashiki J.H. Kobayashi C. Hamamura R. Okabe S. Tauchi T. Ohyashiki K. The oral iron chelator deferasirox represses signaling through the mTOR in myeloid leukemia cells by enhancing expression of REDD1.Cancer Sci. 2009; 100: 970-977Crossref PubMed Scopus (108) Google Scholar]. Because the animal model used for in vivo antitumor effect studies of various ICAs was a mouse or rat model with normal iron status, the results of those studies may be inapplicable to the use of ICAs in patients with SIO. SIO is frequently observed in patients with hematologic malignancies because of multiple transfusions after intensive chemotherapy [13Ruccione K.S. Mudambi K. Sposto R. Fridey J. Ghazarossian S. Freyer D.R. Association of projected transfusional iron burden with treatment intensity in childhood cancer survivors.Pediatr Blood Cancer. 2012; 59: 697-702Crossref PubMed Scopus (25) Google Scholar, 14Armand P. Kim H.T. Rhodes J. et al.Iron overload in patients with acute leukemia or MDS undergoing myeloablative stem cell transplantation.Biol Blood Marrow Transplant. 2011; 17: 852-860Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. We previously presented an SIO mouse model that showed a significant dose-dependent correlation between plasma and liver iron content and histologic evidence of iron overload [15Moon S.N. Han J.W. Hwang H.S. et al.Establishment of secondary iron overloaded mouse model: evaluation of cardiac function and analysis according to iron concentration.Pediatr Cardiol. 2011; 32: 947-952Crossref PubMed Scopus (19) Google Scholar]; therefore, it is necessary to evaluate the effect of ICAs in an SIO animal model. In the present study, the in vitro antileukemic effect of two ICAs (DFO and DFX) on murine leukemia cell lines was evaluated using viability and apoptosis analysis. The two groups of mice, normal and SIO mice, were used for assessing the in vivo antileukemic activity of DFO and DFX. Spleen and bone marrow (femur of BDF1, H-2b/d) mononuclear cells were obtained and maintained in RPMI 1640 with 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA) after ammonium chloride lysis of the red blood cells and straining through a nylon mesh. The total cell number was calculated using a hemocytometer. The murine leukemia cell lines A20 (h-2d, BALB/c origin), L1210 (H-2d, DBA origin), and EL4 (H-2b, C57/BL6 origin) were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI 1640 with 10% fetal bovine serum. DFO and DFX, as ICAs, were provided by Novartis (Basel, Switzerland). The inhibitory effect of DFO and DFX on cell growth was assessed by the Cell Counting Kit-8 (CCK-8, Dojindo Laboratory, Kumamoto, Japan), which measures the ability of viable cells to cleave a tetrazolium salt (WST) to a water-soluble formazan. To determine the effect of DFO and DFX on the viability of leukemia cell lines, two lymphoid leukemia cell lines (EL4 and L1210) were incubated with various concentrations of DFO or DFX. The spleen and marrow cells (1 × 105 cells/well) from mice and leukemia cells (1 × 105 cells/well) from the L1210 and EL4 cell lines were incubated separately in 96-well plates according to the experimental design. After incubation for 48 hours at 37°C, 10 μL of CCK-8 solution was added to each well of the plates. After a 2-hour incubation, the optical density at 450 nm was measured using a microplate reader. Cell viability was expressed as a percentage of the control value, and the inhibitory concentrations (IC50) were calculated. Apoptosis was evaluated for the detection of annexin V–positive and propidium iodide–positive cells by flow cytometry. Briefly, the murine leukemia cells (1 × 106 cells/well) from L1210 and EL4 cell line were incubated in a six-well plate according to the experimental design. After a 48-hour incubation, the cells were washed twice in phosphate-buffered saline (PBS), and annexin V–FITC solution with propidium iodide-PerCP (Roche Diagnostics GmbH, Mannheim, Germany) were added. The prepared cells were then washed twice in PBS, after which FACS Lyse and Fix were added. The analysis of Fas expression was performed on L1210 and A20 leukemia cells according to various concentrations of ICAs. Cells incubated as previously described were washed twice in PBS, and anti–CD95-FITC, and a hamster FITC-conjugated IgG2 (an isotype control) were added. After incubating for 30 min in a dark room, the cells to be tested were washed twice with PBS including 0.04% sodium azide and 5% BSA. The data collection and analysis of flow cytometry were performed using a FACSCalibur flow cytometer (Becton Dickson, Palo Alto, CA, USA). Six-week-old female BDF1 (H-2b/d) mice were purchased from OrientBio (Ga Pyung, Gyeonggi-do, Korea). The mice were adapted to their surroundings for 1 week before commencing the experiments and were housed in temperature- and humidity-controlled rooms with light-dark cycles. All mice were given irradiated food and sterile water ad libitum. These experiments were approved by the Institutional Animal Care and Use committee, Incheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea. The first experiment was performed in non-SIO mice, and the second experiment was conducted in SIO mice. For the second experiment, mice were divided into a placebo control and an SIO group, and both received injections intraperitoneally (i.p.). The SIO group received 300 μL (10 mg) of iron dextran (Sigma-Aldrich, St. Louis, MO, USA) in PBS daily for 5 consecutive days per week (total period of 4 weeks; total dose, 200 mg). The placebo control group received the same volume of PBS. The mice were observed for 2 weeks to monitor their viability after iron loading before the next experiment was performed. Murine leukemia cells (L1210) were harvested, and 2 × 106 cells were injected subcutaneously into the right flank of mice anesthetized with 0.4% Avertin (Sigma-Aldrich). After engraftment, the tumor size was measured three times per week with Vernier calipers. The tumor size (in square millimeters) was calculated using the following formula: Length × Width/2. When tumor mass reached visible size, treatments with ICAs (DFO and DFX) began. DFO (40 mg/kg/day, i.p.) was administered once daily for 6 consecutive days, and DFX (20 mg/kg/day dissolved in 200 μL distilled water, orally) was administered once daily until the cumulative dose reached 300 mg/kg. The mice were observed and weighed daily. In the first experiment, non-SIO mice were divided into a control group (n = 3), a DFO group (n = 5), and a DFX group (n = 5). In the second experiment, the mice were divided into four groups: the SIO mice without ICA treatment (n = 10), the SIO mice treated with DFO (n = 10), the SIO mice treated with DFX (n = 10), and the non-SIO mice with placebo treatment (control group; n = 10). Liver and tumor tissues were collected immediately after death and preserved at −80°C until iron content analysis was performed. The iron content (in milligrams per gram) of the liver and tumor tissues was measured using an atomic absorption spectrophotometer. Comparison of data among the groups was performed according to the nonparametric Mann–Whitney test or repeated measures analysis of variance using SPSS for Windows (version 12.0; SPSS, Chicago, IL, USA). The log-rank method was used in comparing the Kaplan-Meier survival curves. The data were considered statistically significant when p < 0.05. Normal mouse spleen and marrow cells were incubated with various concentrations of DFX and were subsequently evaluated for cell viability. The viability of normal mouse spleen and marrow cells was not changed under various concentrations (6.125–100 μmol/L) of DFX, regardless of the addition of 20 μmol/L ferric chloride (Fig. 1). In EL4 cell cultures, the viability showed a slight dose-dependent decrease in the presence of the ICAs and a marked decrease under high concentrations of the ICAs. The viabilities showed some difference between the two ICAs at high concentrations; the IC50 values were 66.5 μmol/L for DFO and 95.1 μmol/L for DFX (Fig. 2A). The viability of L1210 cells incubated with DFO or DFX showed a marked concentration-dependent decrease, but there was a significant difference between DFO and DFX. The viabilities of L1210 cells showed a more significant decrease with DFX than with DFO at low concentrations. The IC50 values in L1210 cells were 60.9 μmol/L for DFO and 14.7 μmol/L for DFX (Fig. 2B). To determine whether an exogenous iron supply can reverse the cytotoxic effect of the ICAs, stoichiometrically sufficient ferric chloride (0.5 mmol/L) was used. The viabilities of the L1210 cells with the ICAs were not changed despite the addition of ferric chloride. The IC50 values in L1210 cells with ferric chloride were 31.67 μmol/L for DFO and 3.86 μmol/L for DFX (Fig. 2B).Figure 2Viability of EL4 cells and L1210 cells according to concentration of DFO or DFX after incubation for 48 hours. (A) The viability of EL4 cells incubated with DFO or DFX decreased slowly in a dose-dependent manner, and there was some difference between the effects of DFO and DFX on viability at high concentrations. ∗p = 0.03. (B) The viability of L1210 cells incubated with DFX decreased markedly compared to that of cells treated with DFO, especially at low concentrations. Similar trends in viability were observed after saturating DFO or DFX with ferric chloride (FeCl3). The values shown on the plot represent the means ± SD of three independent experiments. ∗p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether apoptosis was induced by DFO or DFX in L1210 cells, we analyzed L1210 cells after DFO and DFX treatment. After 48 hours of exposure to DFO or DFX, the percentage of apoptotic cells increased in a concentration-dependent manner (Fig. 3). To investigate whether chelator-induced apoptosis was related to the Fas pathway, we used flow cytometric analysis of Fas expression using anti-CD95-FITC. The expression of CD95 on L1210 cells did not increase at any concentration of DFX, but increased according to concentration of DFX in A20 cells (Fig. 4). A similar pattern of CD95 expression was found for concentrations of DFO (data not shown).Figure 4Fas expression in A20 cells and L1210 cells evaluated by flow cytometry for the detection of CD95. The expression of CD95 in A20 increased according to concentration of DFX. Fas expression in L1210 did not change despite an increase in DFX concentration.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further study the in vivo antitumor activity of DFO and DFX on tumor growth according to iron control, we used two mouse models (with or without SIO) bearing L1210 leukemic cells. In the first experiment, non-SIO mice were injected subcutaneously with L1210 cells. After 21 days of inoculation, non-SIO mice received PBS alone or ICAs according to the experimental design. The survival of the DFO-treated mice was similar to that of the control mice, whereas the mice treated with DFX showed longer survival than the other groups (p = 0.017; Fig. 5). The initial size difference between tumors was large, preventing a meaningful statistical comparison of final tumor size (data not shown). We performed the second experiment with SIO mice. After 28 days of subcutaneous injection with L1210, the treatment for each mouse group was initiated. The SIO mice treated with DFX showed significantly longer survival than the SIO mice without treatment and the control mice (p < 0.01). The SIO mice treated with DFO showed shorter survival than the control mice (p = 0.016; Fig. 6A). The tumor size was significantly smaller in the SIO mice treated with DFX compared with the SIO mice without treatment and the control mice (p < 0.01). The SIO mice treated with DFO were observed to have a significant decrease in tumor size compared to the SIO mice without treatment and the control mice until the last evaluable day (p < 0.01; Fig. 6B). We measured the iron contents in the liver or tumor of the mice in the second experiment. The liver or tumor iron contents of the mice decreased after ICA treatment (Fig. 7).Figure 6In vivo effects of DFO and DFX in SIO mice and normal control mice inoculated with L1210 cells. (A) The Kaplan-Meier survival curve according to each experimental group. (B) The effect of DFO or DFX on L1210 tumor growth in SIO and normal control mice. The tumor sizes of the four groups were similar until day 33 after L1210 cell inoculation. After day 33, the SIO mice treated with DFX showed a significant decrease in tumor size compared with other groups. The statistical method was a repeated measures analysis of variance to compare the tumor size. The values shown on the plot represent the means ± SD of two independent experiments. ∗p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7The iron content of the liver (A) or the tumor (B) in SIO and normal control mice inoculated with L1210 cells. Liver iron content (LIC) and tumor iron content (TIC) of SIO mice decreased after iron chelation treatment, but remained higher than those of normal control mice. The values shown on the plot represent the means ± SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Patients with hematologic or oncologic disease frequently need to receive red blood cell transfusions, which can lead to serious problems related to SIO [13Ruccione K.S. Mudambi K. Sposto R. Fridey J. Ghazarossian S. Freyer D.R. Association of projected transfusional iron burden with treatment intensity in childhood cancer survivors.Pediatr Blood Cancer. 2012; 59: 697-702Crossref PubMed Scopus (25) Google Scholar, 14Armand P. Kim H.T. Rhodes J. et al.Iron overload in patients with acute leukemia or MDS undergoing myeloablative stem cell transplantation.Biol Blood Marrow Transplant. 2011; 17: 852-860Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Numerous studies have observed that iron provides a favorable environment for neoplastic cell growth [16Huang X. Iron overload and its association with cancer risk in humans: evidence for iron as a carcinogenic metal.Mutat Res. 2003; 533: 153-171Crossref PubMed Scopus (323) Google Scholar]. The antiproliferative and antitumor effects of ICAs have been well recognized in animals with normal iron status; however, the effect of chelators on tumor growth and survival in animals with SIO was previously unclear. In the present study, we showed that ICAs, especially DFX, have an antileukemic effect in EL4 and L1210 leukemia cell lines and in vivo antitumor activity, which contributes to longer survival not only in normal mice but also in SIO mice. In this study, although the IC50 value of ICAs in L1210 cells was lower than that in EL4 cells, ICAs showed a dose-dependent in vitro antileukemic effect on EL4 and L1210 leukemia cells. This effect was not observed in normal murine spleen and marrow cells. These results are similar to those of an earlier report that ICAs induce apoptosis of rapid proliferating cells but not resting cells [17Hileti D. Panayiotidis P. Hoffbrand A.V. Iron chelators induce apoptosis in proliferating cells.Br J Haematol. 1995; 89: 181-187Crossref PubMed Scopus (119) Google Scholar]. In this study, the various concentrations of ICAs showed a significant difference in viability between the two cell lines (EL4 and L1210). The IC50 value of DFO and DFX for EL4 cells was higher than that for L1210 cells. EL4 cells and L1210 cells are derived from murine T cell and B cell leukemia, respectively. Although T cell acute lymphoblastic leukemia (ALL) showed general drug resistance compared with B cell precursor ALL, additional studies will be needed to elucidate the molecular mechanism underlying the different antileukemic effects of ICAs on the two types of murine leukemic cell lines [18Pieters R. den Boer M.L. Durian M. et al.Relation between age, immunophenotype and in vitro drug resistance in 395 children with acute lymphoblastic leukemia–implications for treatment of infants.Leukemia. 1998; 12: 1344-1348Crossref PubMed Scopus (197) Google Scholar]. In this study, the decreased viability of L1210 cells by ICAs could not be recovered, even after saturating ICAs with ferric chloride. Previous studies have shown that iron supplementation can partially or completely reverse the inhibitory effects of ICAs on various cells [5Blatt J. Stitely S. Antineuroblastoma activity of desferoxamine in human cell lines.Cancer Res. 1987; 47: 1749-1750PubMed Google Scholar, 6Becton D.L. Roberts B. Antileukemic effects of deferoxamine on human myeloid leukemia cell lines.Cancer Res. 1989; 49: 4809-4812PubMed Google Scholar, 9Ba Q. Hao M. Huang H. et al.Iron deprivation suppresses hepatocellular carcinoma growth in experimental studies.Clin Cancer Res. 2011; 17: 7625-7633Crossref PubMed Scopus (49) Google Scholar, 10Lescoat G. Chantrel-Groussard K. Pasdeloup N. Nick H. Brissot P. Gaboriau F. Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism.Cell Prolif. 2007; 40: 755-767Crossref PubMed Scopus (38) Google Scholar]. Ferric iron was reported to be less effective in restoring the viability of tumor cells than ferrous iron [19Basset P. Quesneau Y. Zwiller J. Iron-induced L1210 cell growth: Evidence of a transferrin-independent iron transport.Cancer Res. 1986; 46: 1644-1647PubMed Google Scholar, 20Renton F.J. Jeitner T.M. Cell cycle-dependent inhibition of the proliferation of human neural tumor cell lines by iron chelators.Biochem Pharmacol. 1996; 51: 1553-1561Crossref PubMed Scopus (50) Google Scholar]. A possible explanation of this difference is that the added ferric chloride binds to molecules other than ICAs, which prevents Fe3+ from restoring viability. DFX showed a more potent inhibitory effect on L1210 cells than DFO, especially at the low steady-state plasma concentrations of DFO and DFX following the standard dose in human studies [2Kushner J.P. Porter J.P. Olivieri N.F. Secondary iron overload.Hematology Am Soc Hematol Educ Program. 2001; : 47-61Crossref PubMed Scopus (206) Google Scholar, 21Galanello R. Campus S. Origa R. Deferasirox: Pharmacokinetics and clinical experience.Expert Opin Drug Metab Toxicol. 2012; 8: 123-134Crossref PubMed Scopus (25) Google Scholar]. These inhibitory effects of DFO and DFX on L1210 leukemia cells were closely related to apoptosis, because the effects of DFO and DFX on viability of L1210 leukemia cells showed a similar trend to that of the apoptosis-inducing activity of DFO and DFX on L1210 leukemia cells. In this study, the apoptotic effects of DFO and DFX on L1210 leukemia cells agree with the results of other studies indicating that ICAs induce apoptosis in rapid proliferating cells such as myeloid leukemia cells, and hepatoma cells [9Ba Q. Hao M. Huang H. et al.Iron deprivation suppresses hepatocellular carcinoma growth in experimental studies.Clin Cancer Res. 2011; 17: 7625-7633Crossref PubMed Scopus (49) Google Scholar, 10Lescoat G. Chantrel-Groussard K. Pasdeloup N. Nick H. Brissot P. Gaboriau F. Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism.Cell Prolif. 2007; 40: 755-767Crossref PubMed Scopus (38) Google Scholar, 11Kim J.L. Kang H.N. Kang M.H. Yoo Y.A. Kim J.S. Choi C.W. The oral iron chelator deferasirox induces apoptosis in myeloid leukemia cells by targeting caspase.Acta Haematol. 2011; 126: 241-245Crossref PubMed Scopus (21) Google Scholar, 12Ohyashiki J.H. Kobayashi C. Hamamura R. Okabe S. Tauchi T. Ohyashiki K. The oral iron chelator deferasirox represses signaling through the mTOR in myeloid leukemia cells by enhancing expression of REDD1.Cancer Sci. 2009; 100: 970-977Crossref PubMed Scopus (108) Google Scholar]. Our study showed that the ICA-mediated apoptosis of L1210 leukemia cells was not associated with the Fas/FasL pathway. Greene et al. demonstrated that apoptosis induced by ICA tachpyridine in the human cervical cell line HeLa was not prevented by blocking the Fas pathway, but was inhibited by adding Bcl-XL or blocking caspase 9 expression [22Greene B.T. Thorburn J. Willingham M.C. et al.Activation of caspase pathways during iron chelator-mediated apoptosis.J Biol Chem. 2002; 277: 25568-25575Crossref PubMed Scopus (81) Google Scholar]. Kim et al. also reported that DFX induced apoptosis in myeloid leukemia cells through activation of caspase 3, 7, and 9 [11Kim J.L. Kang H.N. Kang M.H. Yoo Y.A. Kim J.S. Choi C.W. The oral iron chelator deferasirox induces apoptosis in myeloid leukemia cells by targeting caspase.Acta Haematol. 2011; 126: 241-245Crossref PubMed Scopus (21) Google Scholar]. Therefore, the ICA-induced apoptosis in L1210 leukemia cells might be associated with the intrinsic rather than the extrinsic pathway for apoptosis, and the molecular mechanism needs further investigation. DFX showed a fourfold lower IC50 value than DFO in the viability analysis of L1210 cells, and a similar trend was observed in the apoptosis test. DFX is one of the synthetic ICAs and has been reported to have fourfold to fivefold greater iron-clearing activity than that of DFO in rats [23Hershko C. Konijn A.M. Nick H.P. Breuer W. Cabantchik Z.I. Link G. ICL670A: A new synthetic oral chelator: evaluation in hypertransfused rats with selective radioiron probes of hepatocellular and reticuloendothelial iron stores and in iron-loaded rat heart cells in culture.Blood. 2001; 97: 1115-1122Crossref PubMed Scopus (131) Google Scholar]. Theoretically, DFX might have advantages over DFO in terms of lipophilicity, because ICAs with high lipophilicity can easily enter cells and deplete intracellular iron [2Kushner J.P. Porter J.P. Olivieri N.F. Secondary iron overload.Hematology Am Soc Hematol Educ Program. 2001; : 47-61Crossref PubMed Scopus (206) Google Scholar, 3Kalinowski D.S. Richardson D.R. The evolution of iron chelators for the treatment of iron overload disease and cancer.Pharmacol Rev. 2005; 57: 547-583Crossref PubMed Scopus (591) Google Scholar]. Previous studies have demonstrated that the antiproliferative activity of ICAs increased in proportion to its hydrophobicity [24Richardson D.R. Tran E.H. Ponka P. The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents.Blood. 1995; 86: 4295-4306Crossref PubMed Google Scholar, 25Hodges Y.K. Antholine W.E. Horwitz L.D. Effect on ribonucleotide reductase of novel lipophilic iron chelators: the desferri-exochelins.Biochem Biophys Res Commun. 2004; 315: 595-598Crossref PubMed Scopus (24) Google Scholar]. In addition to this hydrophobicity, DFX is a tridentate ICA that is kinetically labile and is able to form a partially dissociated iron–ligand complex [3Kalinowski D.S. Richardson D.R. The evolution of iron chelators for the treatment of iron overload disease and cancer.Pharmacol Rev. 2005; 57: 547-583Crossref PubMed Scopus (591) Google Scholar, 4Buss J.L. Torti F.M. Torti S.V. The role of iron chelation in cancer therapy.Curr Med Chem. 2003; 10: 1021-1034Crossref PubMed Scopus (192) Google Scholar]. This property enables DFX to produce reactive oxygen species, leading to cell death or modulation of cellular differentiation [26Chaston T.B. Watts R.N. Yuan J. Richardson D.R. Potent antitumor activity of novel iron chelators derived from di-2-pyridylketone isonicotinoyl hydrazone involves fenton-derived free radical generation.Clin Cancer Res. 2004; 10: 7365-7374Crossref PubMed Scopus (115) Google Scholar, 27Callens C. Coulon S. Naudin J. et al.Targeting iron homeostasis induces cellular differentiation and synergizes with differentiating agents in acute myeloid leukemia.J Exp Med. 2010; 207: 731-750Crossref PubMed Scopus (143) Google Scholar]. To examine the in vivo antileukemic effect of ICAs on L1210 cells according to iron status, the next experiment was performed in both non-SIO mice and SIO mice. In the non-SIO mouse study, the antiproliferative activity of ICAs could not be assessed because of a large variation in tumor size before ICA treatment; however, treatment with DFX in non-SIO mice bearing leukemic cells showed a survival benefit. Studies of the ability of DFO to suppress tumor growth in animals are rare, and their results have been mixed [4Buss J.L. Torti F.M. Torti S.V. The role of iron chelation in cancer therapy.Curr Med Chem. 2003; 10: 1021-1034Crossref PubMed Scopus (192) Google Scholar]. Because DFO is the first approved ICA drug, it has been more convenient to use in clinical trials. Estrov et al. reported that DFO showed an antileukemic effect in infant with neonatal MLL-rearranged leukemia [28Estrov Z. Tawa A. Wang X.H. et al.In vitro and in vivo effects of deferoxamine in neonatal acute leukemia.Blood. 1987; 69: 757-761Crossref PubMed Google Scholar]. The use of DFO in children with neuroblastoma has achieved some success [7Blatt J. Deferoxamine in children with recurrent neuroblastoma.Anticancer Res. 1994; 14: 2109-2112PubMed Google Scholar, 29Donfrancesco A. Deb G. Dominici C. Pileggi D. Castello M.A. Helson L. Effects of a single course of deferoxamine in neuroblastoma patients.Cancer Res. 1990; 50: 4929-4930PubMed Google Scholar], but there is significant room for improvement in the clinical use of ICAs as singular anticancer agents. Despite the antitumor activity of ICAs, their use in patients with normal iron status could create an ethical dilemma. Therefore, our next experiment with SIO mice was performed to investigate whether ICAs have a similar antitumor activity in the SIO condition. The results of the second experiment showed that both DFO and DFX had an antiproliferative effect and that DFX could improve survival in SIO mice. These findings suggest that ICAs, such as DFX, might not eradicate all leukemic cells but can reduce the cancer burden, thus contributing to the survival benefit. Recently, DFO administration after allogeneic hematopoietic stem cell transplantation (HSCT) in patients with SIO was reported to prevent leukemic relapse and improve survival [30Kaloyannidis P. Yannaki E. Sakellari I. et al.The impact of desferrioxamine postallogeneic hematopoietic cell transplantation in relapse incidence and disease-free survival: A retrospective analysis.Transplantation. 2010; 89: 472-479Crossref PubMed Scopus (11) Google Scholar]. SIO during HSCT is associated with adverse outcomes in terms of infection, nonrelapse mortality, early complications (e.g., sinusoidal obstruction syndrome), and late effects (hepatic fibrosis, cardiomyopathy) [31Majhail N.S. Lazarus H.M. Burns L.J. Iron overload in hematopoietic cell transplantation.Bone Marrow Transplant. 2008; 41: 997-1003Crossref PubMed Scopus (105) Google Scholar]. Therefore, the use of ICA in patients with cancer and transfusional iron overload could have the dual benefit of antitumor activity and the decrease of SIO-related complications. In our study, the iron content of the SIO mice treated with ICA was lower than that of the SIO mice without ICA treatment, but still higher than in non-SIO mice. This finding indicates that the antitumor effect of DFX could be associated with iron depletion and another iron-independent molecular mechanism. Recently, a few studies of the molecular mechanism of the antileukemic activity of DFX have been reported. Messa et al. [32Messa E. Carturan S. Maffè C. et al.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.Haematologica. 2010; 95: 1308-1316Crossref PubMed Scopus (109) Google Scholar] demonstrated that DFX inhibits the activity of nuclear factor-κB in myelodysplastic syndrome patients independent of intracellular iron deprivation. Ohyashiki et al. [12Ohyashiki J.H. Kobayashi C. Hamamura R. Okabe S. Tauchi T. Ohyashiki K. The oral iron chelator deferasirox represses signaling through the mTOR in myeloid leukemia cells by enhancing expression of REDD1.Cancer Sci. 2009; 100: 970-977Crossref PubMed Scopus (108) Google Scholar] showed that dephosphorylation of mammalian target of rapamycin (mTOR) in response to DFX, following upregulation of the regulated in development and DNA damage response (REDD1)/tuberous sclerosis complex 2 (TSC2) pathway, induces downregulation of ribosomal S6 protein, thereby inhibiting cell proliferation in myeloid leukemia cells [12Ohyashiki J.H. Kobayashi C. Hamamura R. Okabe S. Tauchi T. Ohyashiki K. The oral iron chelator deferasirox represses signaling through the mTOR in myeloid leukemia cells by enhancing expression of REDD1.Cancer Sci. 2009; 100: 970-977Crossref PubMed Scopus (108) Google Scholar]. Further study will be needed to explore the molecular mechanism of antileukemic effects independent of iron depletion. In this study, we observed that DFO and DFX have an in vitro antileukemic effect and that DFX can suppress tumor growth and improve survival in mice bearing leukemic cells, regardless of their iron status. However, there are some limitations in our study. First, we did not explore the in vitro and in vivo molecular mechanisms underlying the apoptosis of leukemic cells treated with chelators. Second, the cause of early death in the mice treated with DFO was not assessed. because DFO is one of the natural siderophores, a potential complication of its use is the utilization of DFO by other infectious agents to acquire iron [33Marx J.J. Iron and infection: Competition between host and microbes for a precious element.Best Pract Res Clin Haematol. 2002; 15: 411-426Abstract Full Text PDF PubMed Scopus (63) Google Scholar]. Serious infections with Listeria monocytogenes have been reported in SIO mice receiving DFO intraperitoneally [34Ampel N.M. Bejarano G.C. Saavedra Jr., M. Deferoxamine increases the susceptibility of beta-thalassemic, iron-overloaded mice to infection with Listeria monocytogenes.Life Sci. 1992; 50: 1327-1332Crossref PubMed Scopus (16) Google Scholar]. Despite these limitations, our results deserve further study. These results may be the first report to show the in vivo antileukemic effect of DFX in SIO mice bearing B cell origin leukemic cells. The use of DFX might benefit SIO patients with leukemia who need iron chelation and adjuvant antileukemic therapy. This work was partially supported by Novartis Korea. The authors thank Mi-Young Yeom and Eun-Sun Kang for their technical support at Clinical Research Laboratory, Incheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea. No financial interest/relationships with financial interest relating to the topic of this article have been declared.