Deferoxamine Inhibits Acute Lymphoblastic Leukemia Progression through Repression of ROS/HIF-1α, Wnt/β-Catenin, and p38MAPK/ERK Pathways

Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, with a feature of easy to induce multidrug resistance and relapse. Abundant studies have proved that iron overload strengthens the growth and metastasis of tumor cells. Herein, we found that deferoxamine (DFO) effectively decreased the concentration of intracellular iron in ALL cells. DFO inhibited proliferation, induced apoptosis, and obstructed cell cycle of ALL cells, whereas DFO and dextriferron (Dex) used in combination significantly decreased the sensitivity of ALL cells to DFO. Reactive oxygen species (ROS) level was reduced in ALL cells treated with DFO, and the combination of DFO and Dex reversed the effects of DFO. In vivo, DFO inhibited mouse tumor growth. Besides, cyclinD1, β-catenin, c-Myc, hypoxia inducible factor 1 (HIF-1), p-p38MAPK, and p-ERK1/2 protein levels were significantly downregulated, and the levels of prolyl hydroxylase-2 (PHD-2) were upregulated after treated with DFO, whereas Dex treatment reversed those in vivo and in vitro. In conclusion, DFO inhibited the proliferation and ALL xenograft tumor growth, obstructed the cell cycle, and induced apoptosis of ALL cells, probably via inactivating the ROS/HIF-1α, Wnt/β-catenin, and p38MAPK/ERK signaling.


Introduction
Acute lymphocytic leukemia (ALL) is a rare hematological malignancy, contributing to about 20% of new acute leukemias [1]. In the United States, in spite of aggressive firstline treatment, the 5-year overall survival rates of the ALL patients of 40-59-and 60-69-years-old are 24% and 18%, respectively [2]. Recent advances of chemotherapy result in a good chance (approximately 85%) for a cure in children who are newly diagnosed with ALL [3]. However, the remaining 15% of ALL children cannot survive due to multidrug resistance and relapse [4]. erefore, there is an urgent need for new treatment methods to improve the survival rate of ALL patients who have failed treatment or relapsed after the initial response.
Studies have shown that ALL is related to excess iron in the body [5]. Iron is an important nutrient and regulator for physiological processes in cells. Iron can catalyze metabolic activities, which is essential to cell survival and proliferation. According to reports, many forms of cancer are associated with massive iron overload, possibly due to the need for iron as a cofactor to maintain the tumor growth and proliferation [6,7]. Iron deficiency reduced the aggressiveness of a variety of tumor cells, which has important clinical value in the treatment of leukemia [8,9]. Iron chelator triapine has been reported to be able to inhibit the ribonucleotide reductase activity and tumor growth both in vivo and in vitro and has been involved in the phase I and phase II clinical trials [10]. Besides, some other iron-chelating agents also have potential antitumor activity, such as deferasirox [11], DpC [12], Dp44 mT [13], gold tricarboxylic acid (ATA), and deferoxamine (DFO) [14]. According to the reports of Benadiba et al. [15], DFO has significantly affected the survival of T lymphoblastic leukemia/lymphoma cells to induce apoptosis and demonstrated synergistic action with three ALL-specific drugs: L-asparaginase, doxorubicin, and dexamethasone. erefore, as a new type of antitumor drug, iron chelator has attracted wide interest from scholars, but its specific roles and potential mechanism by either disrupting intracellular iron homeostasis or depleting intracellular iron in acute lymphoblastic leukemia have not been fully elucidated. Here, we assessed the tumor-suppressive effect and underlying mechanism of DFO on ALL via targeting iron metabolism. Our findings cast light on the development of more effective treatments for acute lymphoblastic leukemia.

Cell Lines and Cell Culture.
e acute lymphoblastic leukemia cell lines Jurkat and NALM-6 were afforded from the American Type Culture Collection (ATCC, USA) and incubated using the RPMI-1640 (Sigma-Aldrich, Grand Island, NY, USA)) comprised of 10% heat-inactivated fetal bovine serum (FBS; GIBCO, MA, USA) and 50 units/ml penicillin and 500 mg/ml streptomycin (GIBCO, USA). All cells were constantly maintained in a humid atmosphere containing 5% CO 2 at 37°C.

Detection of Intracellular Labile Iron Pool (LIP) Level.
e calcein-AM assay was used to detect the concentration of intracellular iron of ALL cells. Briefly, the NALM-6 and Jurkat cells (2 × 10 5 cells/well) were cultured with or without DFO (100 μmol/L) and dextriferron (Dex, 20 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA). After 24 h, cells were labeled by the final concentration of 0.125 μmol/L calcein-AM (Aladdin Bio-Chem Technology, Shanghai, China) for another 10 min. ese cells were then centrifuged and washed twice to remove unbound fluorescent indicators and resuspended in PBS. Trypan blue with a concentration of 0.25 μg/mL was added to quench the extracellular fluorescence, and the fluorescence intensity of cells was detected by using a fluorescence spectrophotometer.
ereafter, ironchelating agent 2,2-bipyridyl (BIP) with a final concentration of 100 μmol/L was added and treated for 30 min. en, fluorescence intensity of cells was detected by using a fluorescence spectrophotometer and the excitation (Ex) wavelength was set at 495 nm and the emission wavelength (Em) was set at 530 nm. e difference of fluorescence intensity between the two detection results was calculated, which represented the content of intracellular calcein binding iron, namely the intracellular LIP level.

Apoptosis
Analysis. e NALM-6 and Jurkat cells (1 × 10 6 cells/well) were incubated with DFO alone or in combination with Dex for 48 h. After the cells were collected and washed and resuspended with cold PBS, an annexin V-PE/7-ADD apoptosis detection kit (BD Pharmingen ™ , CA, USA) was employed to detect the apoptosis in ALL cells. Eventually, total cell apoptosis was assessed using a Becton-Dickinson FACScan (BD Corporation, USA). Data were analyzed using the E Cell Quest software version 2.0 (BD Corporation, USA).

Cell Cycle Analysis.
e NALM-6 and Jurkat cells (1 × 10 6 cells/well) were incubated with DFO alone or in combination with Dex for 48 h. For the cell cycle assay, the harvested cells were fixed with 75% ethanol at −20°C overnight. e cells were stained with 400 μL of PI (20 μg/ml) and 100 μL of RNase A (100 μg/mL) for 30 min. Cell cycle was detected using flow cytometry and analyzed via the ModFit analysis software (Verity Software House, ME, USA).

Reactive Oxygen Species (ROS) Analysis.
e NALM-6 and Jurkat cells (2 × 10 6 cells/mL) were collected to measure the levels of ROS. DCFH-DA (10 μM) was added into cell suspension, and the cells were incubated for 30 min at 37°C in dark. en, the cells were washed twice with PBS, and the levels of ROS were evaluated on a Becton-Dickinson FACScan flow cytometer at 488 nm.

In Vivo Experiment.
irty BABL/c nude female mice (5 weeks, 18-22 g) were obtained from the Animal Center of the Chinese Academy of Sciences and maintained in a temperature-and light-controlled environment with free access to water and food. NALM-6 cells (4 × 10 6 /200 μL/ mouse) in a serum-free medium were hypodermically injected into the flank of the nude mouse for establishing the acute lymphocytic leukemia models. Mice were distributed randomly into 3 treatment groups and were intraperitoneally given saline, DFO (50 mg/kg/day), and (DFO 50 mg/ kg + Dex 5 mg/kg/d) once a day for 34 days. e tumor weight and volumes were measured and calculated every week. After 35 days, the treated mice were sacrificed and the tumors were dissected and assessed. All mice experiments were performed in compliance with the animal protection legislation and approved by the Animal Experimentation Ethics Committees of Zhengzhou University (approval number: 2018-KY-51).

Western Blot Analysis.
Collected samples were lysed with ice-cold RIPA lysis buffer (Sigma) supplemented with protease inhibitors ( ermo Fisher). e concentration of extracted proteins was quantified by the Braford method (BioRad, Amersham, Sweden). e equal protein lysates 2 Journal of Oncology were denatured in SDS sample buffer and separated by 10% SDS-PAGE followed by electrotransfer onto the nitrocellulose membrane. Membranes were blocked with 5% skimmed milk powder for 30 min and incubated with primary antibodies (against Ki67, Bid, Bcl-xl, GAPDH, β-catenin, cyclinD1, c-Myc, HIF-1α, PHD-2, p38MAPK, p-p38MAPK, p-ERK1/2, and ERK1/2) overnight at 4°C and subsequently hatched with the matching secondary antibodies at room temperature for 2 h. Protein signals were visualized by using ECL reagent and exposed to the ChemiDoc XRS system ( ermo, Bethesda, MD, USA).

Statistical Analysis.
All data were collected from three separate experiments, and values were expressed as mean ± SD. Analyses were conducted using the SPSS 17.0 software. Differences between two groups were evaluated using Student's t-test, and multiple comparisons were conducted by one-way analysis of variance (ANOVA). Differences were statistically significant when the P value < 0.05.

DFO Inhibits Cell Proliferation of NALM-6 and Jurkat
Cells. To evaluate the potential effect of DFO on the proliferation of ALL cells, the CCK-8 assay was also implemented. e proliferation activity of NALM-6 and Jurkat cells subjected to DFO (100 μmol/L) was suppressed in comparison with the control, while the DFO (100 μmol/L) and Dex (20 μg/mL) cotreated group inversed the inhibition effect in comparison to the DFO group (Figures 2(a) and 2(b)). Western blot analyses showed that Ki67 was reduced in the DFO-treated group compared with the control group but increased in the combination group compare to the DFO single-treatment group (Figures 2(c) and 2(d)), which was in concordance with the results of the CCK-8 assay. Moreover, in order to investigate the effects of DFO on the cell cycle of NALM-6 and Jurkat cells, the flow cytometry assay was performed. DFO reduced the number of ALL cells at S stage, while the DFO and Dex cotreated group attenuated the effect (Figures 2(e) and 2(f)). Altogether, DFO suppresses the proliferative ability in ALL cells, whereas Dex attenuated the inhibition.

DFO Promotes Apoptosis in ALL Cells.
Abnormal growth of tumor cells may be closely related to apoptosis [16]. e DFO significantly promoted the apoptosis rates in NALM-6 and Jurkat cells, whereas Dex inhibited the increased apoptosis induced by the DFO treatment compared with the DFO single treatment in ALL cells (Figures 3(a) and 3(b)). e expression of Bid and Bcl-xl was detected by western blot assays to explore the underlying mechanism of DFO on regulating the apoptosis of ALL cells. As shown in Figures 3(c) and 3(d), DFO elevated the levels of Bid and decreased the levels of Bcl-xl in comparison with the control treatment, whereas the DFO and Dex combination treatment reversed the effect of DFO treatment in ALL cells (Figures 3(c) and 3(d)).

DFO Inhibits ROS Production in ALL Cell Lines.
To further explore the effects of DFO on ROS production in ALL cells, flow cytometry assays were performed. As shown in Figures 4(a) and 4(b), the results revealed that DFO treatment significantly reduced the levels of ROS and Dex reversed its effects. Furthermore, western blot results showed that HIF-1α expression was obviously inhibited in ALL cells treated with DFO, and PHD-2 expression was significantly increased, whereas Dex reversed those effects of DFO (Figures 4(c) and 4(d)). ese results suggested that DFO inhibited ROS production by repressing the HIF-1α/PHD-2 pathway in ALL cells. DFO significantly downregulated p-ERK1/2 and p-p38MAPK compared with the control. Moreover, p-p38MAPK and p-ERK1/2 were upregulated in the DFO and Dex cotreated group compare to the DFO-treated group. All results indicated that that DFO exerted antiproliferation, obstructed cell cycle, and induced apoptosis in ALL cells probably involved in the Wnt/β-catenin and p38MAPK/ERK cascades.

DFO Inhibits ALL Xenograft Growth.
To further validate the impact and the underlying mechanism of DFO in vivo, NALM-6 xenograft mice were acquired by hypodermical injection of NALM-6 cells. e xenograft tumor volume and weight in the DFO group were decreased compared to the saline group, while the DFO and Dex combination group significantly reduced the change induced by DFO (Figures 6(a) and 6(b)). Moreover, the western blot assay showed that DFO treatment resulted in a decrease in the protein levels of β-catenin, cyclinD1, c-Myc, p-p38MAPK, and p-ERK1/2 compared to the saline treatment in the tumor tissues, whereas DFO and Dex treatment in combination alleviated the abovementioned observations (Figures 6(c) and 6(d)). Taken together, these results further confirmed that DFO significantly exerted inhibition of ALL via reducing intracellular iron levels in vivo and in vitro.

Discussion
In the United States alone, about 3000 children and teenagers are diagnosed with acute lymphocytic leukemia (ALL) each year [17]. Iron is vital for the physiological function of cells [18]. e abnormal iron metabolism and iron homeostasis disorders have been considered as a vital factor of the pathophysiology of various diseases such as cancer, neurodegenerative disease, cardiovascular complications, and infections [19]. Studies have shown that excessive iron can induce tumor and promote tumor growth, whereas iron deprivation can suppress tumor cell growth and promote apoptosis, and the degree of influence varies with the dose and the effect time of iron deprivation agent [20]. e study by Becton et al. [21] has shown that DFO has antitumor activity against malignant cells of patients with leukemia and acute lymphocytic leukemia, acute myeloid leukemia, and neuroblastoma. DFO is widely used for the treatment of iron overload diseases, and its antitumor effect has attracted more and more attention.
Iron-chelating agents exhibit potential as a new therapy for cancer. In recent years, more and more research studies have been conducted on the antileukemia effect of iron chelators. For example, Chang et al. have reported that deferasirox has strong antileukemia activity [22]. A large number of in vitro, in vivo, and clinical studies reveal that iron-depleting drugs can suppress the aggressiveness of tumor cells and tumor xenografts via chelating intracellular molecules and regulating the levels of iron-regulatory protein and has obvious antiproliferative effects in cancer, which has provided a new direction for anticancer therapies [23,24]. e study by Lee has verified that iron-chelating agents either deferasirox (DFX) or deferoxamine (DFO) could reduce the survival ability of leukemic cells in a dosedependent manner [25]. Interestingly, our results demonstrated that DFO reduced the proliferation of NALM-6 and Jurkat cells and growth of tumor xenografts, whereas Dex attenuated the inhibition, indicating that iron-chelating agent DFO could antagonize iron load and suppress the proliferation and growth of ALL cells and tumor xenografts.
Studies have shown that iron chelation can be used to inhibit the abnormal proliferation of tumor cells for the reason that depletion of iron exhibits antitumor effects [26]. Yang et al. have shown that iron chelator inhibits the proliferation of leukemia cells and induces apoptosis by regulating the expression of apoptosis-related genes [27]. e study of Tomoyasu et al. has exhibited that iron-chelating agent can induce HL-60 and K562 apoptosis and the mechanism is probably by arresting cells growth at the  S-phase [28]. Here, we observed that DFO promoted apoptosis via regulating the expression of proapoptotic Bid and apoptotic suppressor Bcl-xL and obstructed the cell cycle of ALL cell lines, while these effects were further abrogated when the iron levels increased. Iron is reported to be able to result in the abnormal production of ROS in ALL cells [29]. In addition, a reported research has shown that iron promotes the carcinogenesis by inducing the production of ROS through activating JNK and MAPK pathways [30]. In this study, DFO reduced the ROS levels in ALL cells, and the  effects were reversed by Dex, which suggests that iron reduction by DFO confers the inhibitory effects on ROS production. HIF-1α, an important factor, is involved in cell oxidative stress and inflammation [31]. We found that the levels of HIF-1α were reduced in ALL cells treated with DFO, and prolyl hydroxylase domain enzyme-2 (PHD-2) expression was increased, whose effects were reversed by Dex treatment. Our results were consistent with the reported research by Janssens et al. [32]. ey have confirmed that higher levels of HIF-1α are expressed in MDS-MSC of the iron-overload (IO) group than those in the non-iron overload (NIO) group and high levels of ROS induce the HIF-1α level to increase through inhibiting the PHD-2 in MDS-MSC. ese results suggested that iron depletion by DFO inhibited ROS production by repressing the HIF-1α/ PHD-2 pathway in ALL cells. However, the previous study has suggested a reverse effect which is that DFO induces the increase of HIF-1α and DFO-induced leukemic cell apoptosis by mitochondrial pathway-dependent and HIF-1αindependent mechanisms [33].
ese results suggest that DFO-induced apoptosis and DFO-induced inhibition of proliferation and ROS may involve in different initiating signal pathways in different types of cells that remain to be explored. e Wnt/β-catenin signaling regulates the transcription of various oncogenes, including cyclin c-Myc, cyclinD1, and Bcl-w [34]. Abnormal activation of this signaling pathway has been shown in various cancers, such as colorectal cancer, lung cancer, breast cancer, ovary cancer, prostate cancer, synovial sarcoma, and Schwann cell tumors [35]. e widespread loss of control of the Wnt signaling in various malignancies makes it an attractive therapeutic target [36]. Research on targeting Wnt/β-catenin signal transduction in cancers related to Wnt activation has opened up a new avenue for the development of effective drugs to inactivate the Wnt/β-catenin signaling [37].
Abnormal activation of the Wnt/β-catenin signaling correlates with the prognosis of patients suffering acute myeloid leukemia (AML) [38]. In addition, the Wnt/ β-catenin signaling is necessary for the development of highly proliferative leukemia stem cells (LSCs), which is believed to be able to maintain the survival of leukemia [39]. Furthermore, iron load in colorectal cancer cells can promote the Wnt signaling and cell proliferation [40], but iron chelators, including deferoxamine, deferoxamine, and ciclopirox, can inactivate the Wnt signaling, and AML patients taking cyclohexanolamine show a significant decrease in the expression of the Wnt target gene AXIN2. A reported study [41] has shown that depletion of iron weakens the Wnt signaling pathway. Song et al. [42] have reported that acyl compounds inhibit the stability of β-catenin, prevent the transcription induced by Wnt signaling, and further block the growth of cancer cells through binding to iron. Collectively, the protein expression levels of c-Myc, cyclinD1, and β-catenin were lower in DFO-treated cells and tumor xenografts. However, the Dex and DFO cotreated group alleviated the abovementioned observations, suggesting that DFO inhibits the ALL tumor probably through inhibiting the Wnt/β-catenin signaling, which probably serves as a major mechanism in iron chelatormediated cell death of ALL cells by activating downstream apoptotic cascade. Accumulating evidence suggests that iron chelators influence the MAPK cascades, via dephosphorylate MAPKs, such as MAPK/P38 kinase, ERK, and c-Jun N-terminal kinase (JNK) [43], which are at the center of signaling crosstalk of lymphoblastic activation [44]. Lee et al. [45] have demonstrated that DFO can inhibit the growth and promote apoptosis of IHOK and HN4 cells in a time-dependent and dose-dependent manner via activating p38 MAP kinase and ERK, all of which act as a downstream apoptotic cascade that conducts the iron chelator-mediated cell death mechanism. Studies by Kim et al. [46] have reported that p38 MAP kinase and extracellular ERK are activated by chelator DFO at an early stage of incubation. However, the data of Haigl et al. [47] unveil that DFO and hypoxia treatment increases Spry4 expression, accompanied with inhibiting the activation of ERK signaling. Similarly, our current study revealed that  Journal of Oncology DFO markedly reduced the phosphorylation of p38MAPK and ERK, indicating that p38MAPK/ERK arrested the cell cycle and induced apoptosis by iron depletion.

Conclusion
In summary, DFO reduced the proliferation and tumor growth, obstructed the cell cycle and ROS production, and induced apoptosis of ALL cell lines by inactivating the HIF-1α/PHD-2, Wnt/β-catenin, and p38MAPK/ERK pathways by iron depletion. Current results seem likely to generate an interesting adjuvant therapy for the acute lymphocytic leukemia and identify potential targets for anticancer therapy.

Data Availability
All the data generated or analyzed during this study are included within this article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.