CIP1

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Deferasirox protects against hydrogen peroxide-induced cell apoptosis by inhibiting ubiquitination and degradation of p21WAF1/CIP1 Junhua Miao a, Mutao Xu a, Yuhuan Kuang a, Shuhong Pan a, Jianyuan Hou a, Pengxiu Cao a, Xianglin Duan a, Yanzhong Chang a, Habelhah Hasem b, Nan Zhou a, c, Ke Tan a, **, Yumei Fan a, b, * a

Laboratory of Molecular Iron Metabolism, Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, College of Life Science, Hebei Normal University, Shijiazhuang, 050024, PR China Department of Pathology, Carver College of Medicine, The University of Iowa, Iowa City, IA, 52242, United States c Department of Gynecolog, Xingtai People’s Hospital, Xingtai, 054031, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 January 2020 Accepted 27 January 2020 Available online xxx

Deferasirox (DFX) is an iron chelator approved for the treatment of iron overload diseases. However, the role of DFX in oxidative stress-induced cell apoptosis and the exact molecular mechanisms underlying these processes remain poorly understood and require further investigation. In this study, we found that DFX rendered resistant to H2O2-induced apoptosis in HEK293T cells, reduced the intracellular levels of the labile iron pool (LIP) and oxidative stress induced by H2O2. Furthermore, DFX inhibited the ubiquitination and degradation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) via modulation of the interaction of p21 with SCF-Skp2. DFX also showed the inhibition effect on the activation of c-Jun Nterminal kinase (JNK), pro-caspase-3 and related mitochondrial apoptosis pathway induced by H2O2. These results provide novel insights into the molecular mechanism underpinning iron-mediated oxidative stress and apoptosis, and they may represent a promising target for therapeutic interventions in related pathological conditions. © 2020 Elsevier Inc. All rights reserved.

Keywords: Deferasirox Labile iron pool Oxidative stress p21 WAF1/CIP1 Degradation Apoptosis

1. Introduction Hydrogen peroxide (H2O2), a type of reactive oxygen species (ROS), is a central redox signaling molecule [1,2]. It is well known to generate highly reactive and toxic hydroxyl radicals through a Fenton reaction [3]. These hydroxyl radicals are highly reactive and responsible for most oxidative damages to proteins, lipids, sugars, and nucleic acids in biological systems [4]. High concentrations of H2O2 can initiate apoptosis and even necrosis [5,6], and can lead to some pathophysiological conditions such as cancer [7], cataract formation [8], cardiovascular disorders [9], atherosclerosis [10] and neurodegenerative diseases, including Alzheimer’s disease (AD) [11]. Many cellular and animal studies have demonstrated that

Abbreviations: H2O2, hydrogen peroxide; LIP, labile iron pool; DFX, Deferasirox; ROS, Reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; PARP-1, Poly ADP-ribose polymerase 1; JNK, c-Jun N-terminal kinase; MMP, mitochondrial membrane potential. * Corresponding author. No.20 South 2nd Ring Estern Road, Shijiazhuang, Hebei Province, 050024, PR China. ** Corresponding author. E-mail addresses: [email protected] (K. Tan), [email protected] (Y. Fan).

short exposure to H2O2 increases the intracellular labile iron pool (LIP) [12], which plays a critical role in H2O2-induced DNA damage [13]. Deferasirox (DFX) is the first oral iron chelator approved in the United States by the Food and Drug Administration [14]. It has high plasma protein binding properties, in addition to having a long half-life, and it is extensively metabolized in the liver with subsequent fecal excretion [14]. DFX has been used to treat severe chronic iron overload diseases [15,16]. It also shows antiproliferative activity against cancers [17,18]. Yet, the role of DFX in oxidative stress-induced cell damage and apoptosis and the mechanism involved remains elusive and is need of further investigation. The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is an important regulator of cell-cycle progression, cellular responses to DNA damage and cell migration [19]. Another important role of p21 is the protection of cells from apoptosis [20]. p21 can form complex with the apoptosis signal regulating kinase 1 (ASK1) or procaspase-3, and inhibits stress-mediated apoptosis through ASK1/ JNK cascade and mitochondrial apoptosis pathway [21,22]. ROS have been previously shown to trigger p21 protein degradation

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Please cite this article as: J. Miao et al., Deferasirox protects against hydrogen peroxide-induced cell apoptosis by inhibiting ubiquitination and degradation of p21WAF1/CIP1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.155

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through a ubiquitin-dependent pathway [23]. The depletion of p21 has been shown to promote stress-induced apoptosis [24]. In this study, we show that the protective role of DFX in H2O2induced cell apoptosis involved suppressing intracellular LIP and ROS levels, as well as inhibiting ubiquitination and degradation of p21, and subsequent activation of JNK and pro-caspase-3. These findings provide evidence for the development of better-tolerated and more efficient therapies in the treatment of oxidative stressinduced disorders.

fluorescence microscope (OLYMPUS DP80). 2.7. Measurement of intracellular ROS levels

2. Materials and methods

Intracellular ROS levels were measured by detecting the fluorescence intensity of DCF through the DCFH-DAeDCFH-DCF conversion method. Briefly, cells were incubated with DCFH-DA (5 mM) for 30 min at 37
C and then washed with PBS three times to remove free dye. DCF fluorescence was then examined as described previously [25]. An increased value compared to control was viewed as an increase in the intracellular ROS level.

2.1. Cell culture

2.8. Measurement of SOD, GPX and MDA contents

HEK293T cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were grown in Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37
C in a 5% CO2 humidified atmosphere.

The total enzymatic activities of SOD, the content of MDA and reduced GSH were determined in cell lysates using assay kits (Wanleibio) according to the manufacturer’s instructions. Each assay was performed using three biological replicates of each treatment. 2.9. Western blot

2.2. Antibodies and reagents The antibodies for p21 and caspase-3 were purchased from BD Pharmingen (San Diego, CA, USA). A monoclonal antibody against the Flag epitope was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against ubiquitin, PARP, JNK and p-JNK (Thr183/ Tyr185) were obtained from Cell Signaling Technology (Danvers, MA, USA). Polyclonal antibodies against Bcl-2, Bax and Skp2 were obtained from Proteintech (Chicago, IL, USA). All secondary antibodies used for Western blotting and immunofluorescence assays were purchased from KPL (Gaithersburg, MD, USA). An MTT assay kit was purchased from Solarbio (Beijing, China). The calcein-AM, DAPI and DCFH-DA were purchased from Sigma-Aldrich (St. Louis, MO, USA). A PE Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences (San Diego, CA, USA). 2.3. MTT assay The cell viability was determined by MTT assay. The absorbance of each well was measured at 570 nm using an ELx 800 Universal Microplate Reader (Bio-Tek, Inc.).

Western analysis was performed as described [25]. Protein concentrations were measured with a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Aliquots of the extract protein were subjected to SDSePAGE followed by transfer onto polyvinylidene difluoride (PVDF) membranes. Membranes were then blocked with blocking buffer containing 5% nonfat milk and blotted with primary antibodies followed by the corresponding secondary antibodies. The bands were visualized using the SuperSignal West Pico chemioluminescence kit (Pierce). The total density of the protein bands was detected with the LAS4000 System (FujiFilm). 2.10. Reverse transcription-PCR analysis (RT-PCR) Total RNA was extracted with TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. RT-PCR was performed as described previously [25] with the following primers: FTL: sense 50 -atgagctcccagattc-30 , antisense 50 -tccgtcgtgcttgagagtg-30 ; b-actin: sense 50 -agccatgtacgtagccatcc-30 , antisense 50 -tttgatgtcacgcacgattt30 . PCR products were resolved with 1.2% agarose gels and stained with ethidium bromide. b-Actin was used as a housekeeping gene where indicated.

2.4. DAPI staining 2.11. Statistical analysis After treatment, cells were washed with PBS sufficiently, and then fixed with acetic acid/methanol (1:3) solution for 10 min at room temperature and then incubated in DAPI (1 mg/ml) for 5 min. After being washed 3 times with PBS, cells were examined using a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek).

Results are expressed as the mean ± S.E. and ± S.D. Two-way analysis of variance (ANOVA) and Student’s t-testing were applied for comparative analyses. Differences were considered significant for p < 0.05.

2.5. Flow cytometry analysis

3. Results

After treatment, HEK293T cells were collected and then treated with an Annexin V-PE Apoptosis Detection Kit according to manufacturer’s instructions and the DNA content was determined using a fluorescence-activated cell sorting flow cytometer (BD FACS Calibur).

3.1. Effect of DFX on H2O2-induced cell apoptosis

2.6. LIP measurements The LIP of the cells was assessed using the calcein-based method. Briefly, cells were loaded with 50 nM calcein-AM for 30 min at 37
C and then washed with PBS three times to remove free dye. The baseline of calcein fluorescence was examined using a

Previous study showed that DFX was markedly effective at preventing t-BHP-induced oxidative injury to cells [26]. DFX treatment also improved iron-mediated oxidative DNA damage in patients with transfusion-dependent myelodysplastic syndrome [27]. To explore the protective effects of DFX against cell apoptosis induced by H2O2, HEK293T cell viability and apoptosis were determined using MTT assay, DAPI staining and flow cytometry analysis. Results revealed that after treatment with 500 mM H2O2 for 24 h, cell viability was significantly decreased to 15% as compared to the control group, whereas 30 min pretreatment with

Please cite this article as: J. Miao et al., Deferasirox protects against hydrogen peroxide-induced cell apoptosis by inhibiting ubiquitination and degradation of p21WAF1/CIP1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.155

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Fig. 1. DFX suppresses H2O2-induced cell apoptosis. (A) HEK293T cells were treated with 500 mM H2O2 for 24 h in the absence or presence of 0e100 mM DFX (0.5 h pretreatment). Cell viability was determined using an MTT assay. Data is shown as the mean ± SEM (n ¼ 5). *p < 0.05, ***p < 0.001, compared with PBS-treated cells; &&p < 0.01, &&& p < 0.001, compared with H2O2-treated cells. (B, C) HEK293T cells were treated with 500 mM H2O2 for 24 h in the absence or presence of 50 mM DFX pretreatment (30 min). The chromatin

Please cite this article as: J. Miao et al., Deferasirox protects against hydrogen peroxide-induced cell apoptosis by inhibiting ubiquitination and degradation of p21WAF1/CIP1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.155

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Fig. 2. DFX inhibits H2O2-induced upregulation of intracellular LIP and oxidative stress in HEK293T cells. HEK293T cells were pretreated with or without 50 mM DFX for 30 min before exposure to 500 mM H2O2 for 2 h. After that, (A) the cellular LIP levels were assessed by microscopic analysis following calcein fluorescence staining; (B) intracellular ROS generation was monitored via DCF fluorescence intensity; (C) MDA content was determined by the thiobarbituric acid (TBA)-based colorimetric method; (D) Reduced GSH was measured with a glutathione reductase/5,50 -dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay kit; (E) Total SOD activities were measured using the hydroxylamine method. Data are shown as the mean ± SEM (n ¼ 3) of three experiments. **p < 0.01, ***p < 0.001, compared with PBS-treated cells; &&p < 0.01, &&& p < 0.001, compared with H2O2-treated cells.

10e100 mM DFX restored cell viability in a dose-dependent manner (Fig. 1A). Exposure of cells to H2O2 (500 mM) for 24 h increased condensation of the nuclear chromatin compared with the control group as detected by DAPI staining, which was decreased significantly by DFX pretreated (Fig. 1B). Flow cytometry analysis revealed that the ratio of apoptotic cells (LR þ UR) rose to 86% by treatment of cells with H2O2 for 24 h. Pretreatment with DFX decreased the percentage of apoptotic cells to 7% (Fig. 1C).

3.2. Effect of DFX on the H2O2-induced increase of intracellular LIP and oxidative stress Previous reports have demonstrated that H2O2 treatment substantially increased the cytosolic LIP level in Jurkat T J16 cells and the neuroblastoma cell line SH-SY5Y [12,28]. In order to investigate the effects of DFX on cytosolic LIP and oxidative state under oxidative stress conditions in HEK293T cells, LIP and ROS were detected with the calcein-fluorescence assay and DCF fluorescence, respectively. As shown in Fig. 2A, 500 mM H2O2 treatment greatly reduced intracellular calcein fluorescence, indicating an increase in LIP compared with control cells. Pretreatment with DFX (50 mM) prevented this H2O2-induced calcein fluorescence quenching, suggesting that iron chelation with DFX suppressed an H2O2induced increase in LIP in HEK293T cells (Fig. 2A). Furthermore, H2O2 significantly increased DCF fluorescence, indicating an increase in ROS compared to control cells. Pretreatment with DFX (50 mM) protected against H2O2-induced ROS formation (Fig. 2B). Overproduction of ROS causes oxidative damage, along with increased MDA levels, GSH depletion and decreased SOD enzyme activity. Therefore, we evaluated whether the protective effects of DFX were also associated with MDA, GSH and SOD. As shown in Fig. 2CeE, after the treatment of cells with 500 mM H2O2 for 2 h, the

level of MDA increased nearly seven-folds (Fig. 2C), the content of reduced GSH was decreased by 75% (Fig. 2D), and the enzyme activity of total SOD (T-SOD) was decreased by 50% (Fig. 2E) compared with the control group in HEK293T cells, respectively. Pretreatment with DFX (50 mM) markedly inhibited the increase in MDA level, restored the reduction in GSH level and total SOD activity as well. These data suggest that the H2O2-induced oxidative stress in HEK293Tcells was a result of an increase in LIP and indicated that DFX inhibited H2O2induced oxidative stress through iron sequestration.

3.3. Effect of DFX on H2O2-induced ubiquitin-dependent degradation of p21 p21 has been found to correlate with cell cycle regulation, DNA repair, and modulation of apoptosis [19]. ROS were previously shown to trigger p21 protein degradation through a proteasome and ubiquitin-dependent pathway [23]. Otherwise, ROS-mediated p21 downregulation has been shown to promote apoptosis [24,29]. To determine the effect of DFX on the H2O2-induced p21 protein degradation, HEK293T cells were incubated with medium alone or medium containing 500 mM H2O2 in the presence or absence of 50 mM DFX. These results showed that DFX prevented the H2O2-induced decrease in the p21 protein level (Fig. 3A). Next, we co-transfected HEK293T cells with a plasmid encoding Flag-p21 and HA-ubiquitin (HA-UB). Cells were pretreated with 20 mM MG132 to prevent the degradation of ubiquitin-conjugated p21. Flagp21 protein was immunoprecipitated with an anti-Flag antibody after incubation in medium alone or medium containing 500 mM H2O2 in the presence or absence of 50 mM DFX for 4 h. Western blot analysis was then performed using anti-Flag or antiubiquitin (UB) antibodies (Fig. 3B). Results showed that H2O2 led to a significant increase in p21 polyubiquitylation, whereas the

Please cite this article as: J. Miao et al., Deferasirox protects against hydrogen peroxide-induced cell apoptosis by inhibiting ubiquitination and degradation of p21WAF1/CIP1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.155

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Fig. 3. DFX decreases H2O2-induced ubiquitin-dependent degradation of p21. (A) HEK293T cells were treated with 500 mM H2O2 for 4 h in the absence or presence of 50 mM DFX. The p21 protein level was determined by Western blot. b-actin was used to ensure equal protein loading. (B) HEK293T cells were transiently transfected with Flag-p21 and HA-UB. 24 h after transfection the cells were treated with 500 mM H2O2 for 4 h in the absence or presence of 50 mM DFX. The proteasome inhibitor MG-132 (20 mM) was added as indicated. Cell lysates were immunoprecipitated using an anti-Flag antibody and then immunoblotted with an anti-UB or anti-Flag antibody. (C) HEK293T cells were transiently transfected with Flag-p21. 24 h after transfection the cells were incubated 500 mM H2O2 for 4 h in the absence or presence of 50 mM DFX. Cell lysates were immunoprecipitated using an antiFlag antibody and then immunoblotted with an anti-Skp2 antibody. The experiments were repeated three times, and similar results were obtained.

incubation with DFX reduced the levels of polyubiquitylated p21. This indicated that DFX blocked p21 proteasomal degradation via decreasing p21 polyubiquitylation induced by H2O2.

The E3 ubiquitin ligase complex SCF-Skp2, which contains Skp2 (S phase kinase-associated protein 2), has been reported to induce p21 ubiquitylation and degradation [23]. To assess the effect of DFX

condensation was visualized by DAPI staining (B) and apoptosis was detected by PE Annexin V staining and flow cytometry analysis (C). Experiments were repeated three times and similar results were obtained. Data are shown as mean ± S.D. ***p < 0.001, compared with PBS-treated cells; &&&p < 0.001, compared with H2O2-treated cells.

Please cite this article as: J. Miao et al., Deferasirox protects against hydrogen peroxide-induced cell apoptosis by inhibiting ubiquitination and degradation of p21WAF1/CIP1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.155

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Fig. 4. DFX inhibits H2O2-induced the activation of JNK, pro-caspase-3, and related apoptosis pathway. (A) HEK293T cells were treated with 500 mM H2O2 for 120 min in the absence or presence of 50 mM DFX pretreatment (30 min). The phosphorylation of JNK and total JNK proteins was determined by Western blot analysis. ***p < 0.001, compared with PBS-treated cells; &&& p < 0.001, compared with H2O2-treated cells. (B) HEK293T cells were treated with 500 mM H2O2 for 6 h in the absence or presence of 50 mM DFX pretreatment (30 min). The MMP changes were determined by Mitochondrial Membrane Potential Assay Kit. (C) HEK293T cells were treated with 500 mM H2O2 for 24 h in the absence or presence of 50 mM DFX pretreatment (30 min). The protein levels of Bcl-2, Bax, pro-caspase-3 and cleaved caspase-3 were determined by Western blot analysis. ***p < 0.001, compared with PBS-treated cells; &&& p < 0.001, compared with H2O2-treated cells. All experiments were repeated three times and similar results were obtained.

on Skp2p21 interactions, Flag-p21 plasmid was transfected into HEK293T cells, and immunoprecipitation was performed using an anti-Flag antibody. We found that the amount of Skp2 binding to p21 was significantly increased after treatment with 500 mM H2O2 for 4 h. Pretreatment with 50 mM DFX decreased the interaction between p21 and Skp2 (Fig. 3C), suggesting that DFX decreased the ubiquitin dependent degradation of p21 by blocking the interaction

between p21 and Skp2. 3.4. Effect of DFX on H2O2-induced activation of JNK, pro-caspase-3 and mitochondrial apoptosis pathway Previous report showed that p21 acts as an inhibitor of apoptosis by binding with ASK1, and inhibits the activation of the SAPK/JNK

Please cite this article as: J. Miao et al., Deferasirox protects against hydrogen peroxide-induced cell apoptosis by inhibiting ubiquitination and degradation of p21WAF1/CIP1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.155

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apoptosis pathway [21,30]. Furthermore, it also forms complex with pro-caspase-3 to prevent its activation and inhibit the main mitochondrial pathway of apoptosis induction [31,32]. To examine the effect of DFX on the activation of JNK, HEK293T cells were treated with 500 mM H2O2 for 120 min in the presence or absence of 50 mM DFX. As shown in Fig. 4A, the phosphorylation of JNK was increased significantly after H2O2 treatment, and this phosphorylation was suppressed by pre-treating the cells with DFX. Next, we found that a significant decrease in the mitochondrial membrane potential (MMP) occurred after 500 mM H2O2 treatment for 6 h (Fig. 4B). Pretreatment with DFX (50 mM) restored the depolarization of the MMP. Furthermore, the anti-apoptotic Bcl-2 protein level was decreased, the pro-apoptotic Bax and the cleaved caspase-3 were increased after H2O2 (500 mM, 24h) treatment. Pretreatment with DFX (50 mM) restored the expression of Bcl-2, blocked the expression of Bax and the cleavage of caspase-3 (Fig. 4C). These results demonstrated that DFX can stabilize p21 protein to inhibit the activation of ASK1/JNK, pro-caspase-3 and related apoptosis pathway to protect against the apoptosis induced by H2O2. 4. Discussion Maintaining the redox balance in cells is important for normal cellular physiology and healthy organisms. Excess production of ROS in cells results in increased oxidative stress and can cause pathological conditions [4]. H2O2, a nonpolar molecule, is known to exert cytotoxic effects on cells and tissues by generating hydroxyl radicals with free iron from the intracellular LIP via Fenton reaction [3]. Iron (Fe) is essential for cellular metabolism, proliferation and differentiation [33,34]. It also catalyzes the decomposition of hydrogen peroxide into free radicals such as hydroxyl and hydroperoxyl radicals. Recent publications by others have shown that the iron chelator DFO significantly protects against cell death induced by H2O2 via labile iron chelation in J16 and Jurkat cells [12,35,36], suggesting the critical role of iron in H2O2-induced cell death. However, the exact molecular mechanisms underlying these processes remain poorly understood. In this study, we demonstrated that the long half-life oral iron chelator DFX protects cells from H2O2-induced apoptosis by suppressing cellular LIP changes, oxidative stress, as well as by blocking p21 proteasomal degradation via decreasing p21 polyubiquitylation. In previous studies, DFX has been shown to have many beneficial effects in iron overload diseases, such as cancer, and so on [16,17]. This study, however, was focused on its antiapoptotic effects and mechanism. The study presented here showed that DFX effectively inhibits H2O2-induced apoptosis (Fig. 1). It is most likely that DFX suppresses apoptosis by regulation of cellular LIP and oxidative stress status (Fig. 2). In line with this, treatment of cells with ferric citrate, an iron supplier, dramatically increased H2O2-induced cellular LIP, oxidative stress and apoptosis (data not shown). The p21 gene was identified as a cell-cycle progression inhibitor by binding to cyclin-dependent kinase (CDK). Previous studies have indicated another important role for p21 as an inhibitor of apoptosis [19]. Cytoplasmatic p21 protein is able to bind to procaspase-3, prevent its activation, and inhibit the main mitochondrial pathway of apoptosis induction [31,32]. The anti-apoptotic role of p21 is further supported by the ability of p21 to bind and inhibit pro-apoptotic kinases such as apoptosis signal-regulating kinase 1 (ASK1), as well as inhibiting the activation of the SAPK/ JNK apoptosis pathway [29,30]. ROS were previously shown to trigger p21 protein degradation through a proteasome and ubiquitin-dependent pathway [23]. p21 is sensitive to redox status, and the direct interaction between p21 and Skp2 was shown to be

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elevated in H2O2-treated cells [23]. p21 depletion also increased dexamethasone-induced cell death and ROS generation [24]. In this study, we found that H2O2 could induce p21 degradation by the proteasome in a ubiquitination-dependent manner in HEK293T cells. DFX restored the H2O2-induced decrease of p21 protein levels and increased p21 stability by blocking p21Skp2 interaction and subsequent p21 ubiquitination (Fig. 3). We also found the significant inhibition effect of DFX on the H2O2-induced activation of JNK and pro-caspase-3 (Fig. 4). Consistent with these results, DFX significantly inhibited H2O2-induced MMP changes, downregulated the protein expression of Bax, which is a critical executor in the mitochondrial apoptotic process, while DFX promoted the protein expression of Bcl-2, which is an important anti-apoptotic protein (Fig. 4). In summary, our study demonstrated novel mechanisms by which DFX inhibits H2O2-induced cell cytotoxicity and apoptosis. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by grants from the key projects of Hebei science and technology research projects for colleges and universities in Hebei (ZD2016080), key projects of Hebei Normal University (L2018Z07), the National Natural Science Foundation of China (31701006), the One Hundred Person Project of Hebei Province (E2016100019), the Natural Science Foundation of Hebei Province (C2017205129). No competing financial interests exist for any of the authors. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. References [1] E.A. Veal, A.M. Day, B.A. Morgan, Hydrogen peroxide sensing and signaling, Mol. Cell 26 (2007) 1e14. [2] G. Bartosz, Reactive oxygen species: destroyers or messengers? Biochem. Pharmacol. 77 (2009) 1303e1315. [3] A. Fischbacher, C. von Sonntag, T.C. Schmidt, Hydroxyl radical yields in the Fenton process under various pH, ligand concentrations and hydrogen peroxide/Fe(II) ratios, Chemosphere 182 (2017) 738e744. [4] S. Saeidnia, M. Abdollahi, Toxicological and pharmacological concerns on oxidative stress and related diseases, Toxicol. Appl. Pharmacol. 273 (2013) 442e455. [5] M. Abdollahi, S.V. Shetab-Boushehri, Is it right to look for anti-cancer drugs amongst compounds having antioxidant effect? Daru 20 (2012) 61. [6] M. Abdollahi, M. Salehnia, S. Salehpour, et al., Human ovarian tissue vitrification/warming has minor effect on the expression of apoptosis-related genes, Iran. Biomed. J. 17 (2013) 179e186. [7] R.F. Dielschneider, E.S. Henson, S.B. Gibson, Lysosomes as oxidative targets for cancer therapy, Oxid. Med. Cell Longev. 2017 (2017) 3749157. [8] X.H. Jin, K. Ohgami, K. Shiratori, et al., Inhibition of nuclear factor-kappa B activation attenuates hydrogen peroxide-induced cytotoxicity in human lens epithelial cells, Br. J. Ophthalmol. 91 (2007) 369e371. [9] K. Sugamura, J.F. Keaney Jr., Reactive oxygen species in cardiovascular disease, Free Radic. Biol. Med. 51 (2011) 978e992. [10] J.E. Klaunig, L.M. Kamendulis, B.A. Hocevar, Oxidative stress and oxidative damage in carcinogenesis, Toxicol. Pathol. 38 (2010) 96e109. [11] J.A. Klein, S.L. Ackerman, Oxidative stress, cell cycle, and neurodegeneration, J. Clin. Invest. 111 (2003) 785e793. [12] A. Al-Qenaei, A. Yiakouvaki, O. Reelfs, et al., Role of intracellular labile iron, ferritin, and antioxidant defence in resistance of chronically adapted Jurkat T cells to hydrogen peroxide, Free Radic. Biol. Med. 68 (2014) 87e100. [13] A. Barbouti, P.T. Doulias, B.Z. Zhu, et al., Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage, Free Radic. Biol. Med. 31 (2001) 490e498. [14] H.H. Chang, M.Y. Lu, S.S. Peng, et al., The long-term efficacy and tolerability of oral deferasirox for patients with transfusion-dependent beta-thalassemia in Taiwan, Ann. Hematol. 94 (2015) 1945e1952.

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Please cite this article as: J. Miao et al., Deferasirox protects against hydrogen peroxide-induced cell apoptosis by inhibiting ubiquitination and degradation of p21WAF1/CIP1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.155