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Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes Xiaoxue Yu a, b, Yang Ruan c, Xiuqing Huang b, Lin Dou b, Ming Lan b, Ju Cui b, Beidong Chen b, Huan Gong b, Que Wang a, b, Mingjing Yan a, b, Shenghui Sun b, Quan Qiu b, Xiyue Zhang b, Yong Man b, Weiqing Tang b, Jian Li a, b, Tao Shen a, b, * a
Peking University Fifth School of Clinical Medicine, Beijing, 100730, China The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, P.R. China, Beijing, 100730, China c Beijing Tiantan Hospital, Capital Medical University, 100070, Beijing, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 November 2019 Accepted 5 December 2019 Available online xxx
Doxorubicin, as a first line chemotherapeutic agent, its usage is limited owing to cardiotoxicity. Necroptosis is a new form of programmed cell death, and recent investigations indicated that necroptosis is vitally involved in serious cardiac pathological conditions. Dexrazoxane is the only cardiac protective drug approved by FDA for anthracycline. We aimed to explore whether and how dexrazoxane regulates doxorubicin-induced cardiomyocyte necroptosis. First, doxorubicin could cause heart failure and reduce cardiomyocyte viability by promoting cell apoptosis and necroptosis in vivo and in vitro. Second, necroptosis plays an important role in doxorubicin induced cardiomyocyte injury, which could be inhibited by Nec-1. Third, dexrazoxane increased cell viability and protect heart function by decreasing both cardiomyocyte apoptosis and necroptosis after doxorubicin treatment. Forth, dexrazoxane attenuated doxorubicin-induced inflammation and necroptosis by the inhibition of p38MAPK/NF-kB pathways. These results indicated that dexrazoxane ameliorates cardiotoxicity and protects heart function by attenuating both apoptosis and necroptosis in doxorubicin induced cardiomyocyte injury. © 2019 Elsevier Inc. All rights reserved.
Keywords: Dexrazoxane Doxorubicin Necroptosis Apoptosis p38MAPK NF-kB
1. Introduction Cancer is the second leading cause of death worldwide [1]. Doxorubicin (DOX), also known as adriamycin, is an anthracycline antibiotic derived from Streptomyces [2]. Doxorubicin alone or in combination with other chemotherapy has been a common first line therapy for a wide range of cancers, such as lymphoma, leukemia, solid tumor (such as breast cancer and colon cancer) [3e5]. Unfortunately, its clinical usage is hampered by its adverse side effects, most notably a time and dose-dependent cardiotoxicity, which may ultimately result in early or late onset irreversible degenerative cardiomyopathy and congestive heart failure [6]. One of the crucial pathological factors of heart failure is the loss of terminally differentiated cardiomyocytes [7]. Several mechanisms modulating doxorubicin-induced cardiotoxicity have been studied
* Corresponding author. Peking University Fifth School of Clinical Medicine, Beijing, 100730, China. E-mail address: [email protected] (T. Shen).
including apoptosis, dysregulated autophagy, and necrosis [8e10]. Necroptosis is a new form of programmed cell death, which is different from traditional apoptosis and necrosis. Necroptosis is a kind of caspase-independent cell death, mainly mediated by RIPK (receptor interaction protein kinase) and MLKL (mixed lineage kinase domain-like protein) [11]. Necroptosis can trigger inflammation, and plays a significant role in pathological process of various diseases. Recent investigations indicated that necroptosis is vitally involved in serious cardiac pathological conditions, including ischemia-reperfusion injury, myocardial infarction and heart failure [12]. A growing body of evidences confirm that doxorubicin can induce necroptosis in various cancer cell lines [13,14] Previous research also suggests that RIP3 deficiency blocks doxorubicininduced myocardial necroptosis in mice [12]. However, the role of necroptosis in doxorubicin-induced cardiotoxicity remains to be elucidated. Dexrazoxane (DEX) is a water-soluble ring-closed analogue of the iron chelator ethylenediaminetetraacetic acid (EDTA), which belongs to the bisdioxopiperazine class of compounds [15].
https://doi.org/10.1016/j.bbrc.2019.12.027 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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Dexrazoxane is the only FDA approved cardioprotective drug coadministered with anthracycline for preventing anthracyclineinduced cardiac damage [6]. Current studies show that dexrazoxane can reduce oxidative stress by means of chelating free iron and prevent anthracycline from binding to the topisomerase 2b complex [16]. The precise cardioprotective mechanism of dexrazoxane is not fully understood, and it is still unclear whether and how dexrazoxane regulates doxorubicin-induced injury in cardiomyocyte. In the present study, we established a doxorubicin-induced mouse heart failure model in vivo and cardiomyocytes injury model to determine the protective mechanism of dexrazoxane on cardiomyocytes, which may offer new evidence for the cardioprotective mechanisms of dexrazoxane.
deoxyuridine (Sigma-Aldrich, USA) and incubated at 37 C in a humidified atmosphere with 5% CO2 and 95% air (v/v). 2.5. Preparation of drugs Doxorubicin hydrochloride was purchased from the Cell signaling Technology (Beverly, MA, US). The doxorubicin hydrochloride was dissolved in deionized water to 5 mM and then diluted with DMEM culture medium containing 1% FBS at different concentrations. Dexrazoxane (DEX) and Nec-1 were purchased from the Sigma-Aldrich, USA, DEX and Nec-1 were separately dissolved in DMSO to 60 mM and 10 mM, then diluted them to different concentrations with DMEM culture medium containing 1% FBS. 2.6. Cell viability and lactate dehydrogenase (LDH) leakage assays
2. Materials and methods 2.1. Animals Ten-week-old male C57BL/6 mice were provided by SPF (Beijing) Biotechnology Co. The mice (n ¼ 40) were randomly divided into 4 groups. Control (Con) mice were injected 0.1 ml of 0.167 mol/ L sodium lactate solution and saline as placebo intraperitoneally. Doxorubicin-treated mice (DOX) were injected with doxorubicin hydrochloride dissolved in normal saline (10 mg/kg i.p.) three times at day 1, 3 and 5, and 0.1 ml of 0.167 mol/L sodium lactate solution 3 h before DOX treatment. Doxorubicin plus Dexrazoxane treatment mice (DOX þ DEX) were pretreated with 0.1 ml DEX solution (200 mg/kg/day, dissolved in 0.167 mol/L sodium lactate solution) 3 h before 10 mg/kg DOX treatment for three times at day 1, 3 and 5 intraperitoneally. Dexrazoxane-treated mice (DEX) were injected 0.1 ml DEX solution. The dosage of DOX and DEX was modified according to previous studies [17]. All of the mice in the 4 groups were euthanized 7 days after the initial injection of doxorubicin. All animal experiments conformed to the protocols approved by Beijing Hospital Animal Use and Care Committee, and the Guide for Care and Use of Laboratory Animals (NIH Publication # 85-23, revised 1996). 2.2. Echocardiography Mice were lightly anesthetized with 1e1.5% isoflurane in oxygen until the heart rate stabilized to 400 to 500 beats per minute. Echocardiography was performed using Vevo 770 and Vevo 2100 (VisualSonics) instruments. Fraction shortening (FS), ejection fraction (EF), left ventricular internal diameter (LVID) during systole, LVID during diastole, end-systolic volume, and end-diastolic volume were calculated with Vevo Analysis software (version 2.2.3) as previously described [8]. After echocardiography measurements, mice were euthanatized by cervical dislocation, and the hearts were collected for further analyses. 2.3. DNA electrophoresis assay DNA electrophoresis assay was performed as previously described [18]. 2.4. Neonatal mouse ventricular myocyte isolation and culture Neonatal mouse ventricular myocytes (NMVMs) were isolated from 1-3-day-old C57BL/6J mice by digestion with the combination of trypsin and collagenase type II [19]. Cardiomyocytes were plated at a density of 6.6 104 cells/cm2 in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA) in the presence of 0.1 mM 5-bromo-2-
Cells (5 103 cells/well) were seeded in 96-well plates, the cells were divided into four groups: Con groups were treated with DMSO alone as control, DOX group were treated with 0.5 mM of doxorubicin hydrochloride, DOX þ DEX group were pretreated with 200 mM dexrazoxane 4 h before 0.5 mM of doxorubicin hydrochloride treatment, and DEX group were treated with 200 mM dexrazoxane. Then the cells viability was determined with MTT assay kit (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-aldrich, US) according to the manufacturer’s protocol. The LDH concentration was detected using an LDH assay kit according to the manufacturer’s protocol (Solarbio, Beijing, China). The absorbance was obtained by a spectrophotometer. All assays were performed at least three times. 2.7. Western blot analysis Western blotting was performed as previously described [20]. 2.8. Statistical analysis Values are presented as the means ± SEM of at least three experiments. Data were analyzed using GraphPad Prism 6.0 statistical software (GraphPad Software, CA, USA). The differences between two groups were analyzed using Student’s t-test, and multiple comparisons were analyzed using one-way ANOVA with Bonferroni’s correction. For all tests, probability value less than 0.05 was defined as statistically significant. 3. Results 3.1. Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity and protects heart function in vivo To determine whether dexrazoxane could protect heart function after doxorubicin treatment in vivo, we generated a heart failure model using doxorubicin-treated male C57BL/6J mice. Lower body weight and higher heart/body weight ration were observed in control mice than doxorubicin-treated mice, which could be improved by dexrazoxane. (Fig. 1A and B). Meanwhile, we measured heart function by Echo analysis. Compared with control mice, doxorubicin-treated mice exhibited decreased heart function as measured by the ejection fraction (EF) % and fraction shortening index (FS) % upon echocardiogram. However, pretreatment with DEX significantly preserved heart function in vivo (Fig. 1CeE). Moreover, Western blot analysis revealed that DEX suppressed doxorubicin-induced RIP1, RIP3 MLKL and cleaved-caspase 3 (active form of caspase-3) expression in mouse heart (Fig. 1FeJ). These results suggested that doxorubicin could cause cardiotoxicity and heart function loss by promoting both apoptosis
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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Fig. 1. Dexrazoxane ameliorates doxorubicin-caused necroptosis and apoptosis in vivo. (AeB) Body weight and ratio of heart weight to body weight in Control mouse (Con), doxorubicin treated mouse (DOX), doxorubicin and dexrazoxane pretreated mouse (DOX þ DEX), dexrazoxane treated mouse (DEX). (CeE) Echocardiography of Con, DOX, DOX þ DEX, DEX mouse heart function 7 days after fist treatment as shown by fractional shortening % (FS %) and ejection fraction % (EF %) (n ¼ 6, *p < 0.05, **p < 0.01). (FeJ) Western blotting and average data for RIP1, RIP3, MLKL and cleaved caspase 3 in Con, DOX, DOX þ DEX, DEX groups 7 days after fist treatment (n ¼ 5, *p < 0.05, **p < 0.01).
and necroptosis. Dexrazoxane treatment remarkably alleviated the cardiotoxicity and the heart function loss in vivo.
3.2. Doxorubicin induces cardiotoxicity by promoting both apoptosis and necroptosis in cardiomyocytes Cell viability assay was used to determine the viability of neonatal mouse ventricular myocytes (NMVMs) after doxorubicin treatment. We found that the cell activity decreased with the increase of doxorubicin concentration in a concentration-dependent manner (Fig. 2A). Moreover, in order to reveal the effects of doxorubicin treatment on different kinds of cell death type, we used DNA electrophoresis assay to analyze cell death type. DNA electrophoresis shows that both DNA smear and DNA laddering appeared at the 0.5 mM of DOX treatment, suggesting both necrosis and apoptosis existed in the doxorubicin treated cardiomyocytes. When increased the doxorubicin concentration to 1e3 mM, there is only DNA smear in the cardiomyocyte, which suggests that high concentration of DOX leads to classic necrosis (Fig. 2B). Further, we analyzed cell apoptosis and necroptosis in doxorubicin treated cardiomyocytes. Necroptosis related proteins such as RIP1, RIP3,
MLKL and apoptosis related cleaved caspase-3 were detected by Western blotting. Our data manifested that both RIP1, RIP3 and MLKL were significantly upregulated when exposed to 0.5 mM doxorubicin treatment compared with the control. While cleaved caspase-3 (active form of caspase-3) increased obviously at the doxorubicin concentration of 1 mMe3 mM (Fig. 2CeG). All these results suggest that low concentration of doxorubicin (0.5 mM) could induce cardiotoxicity by increasing both cell apoptosis and cell necrosis in cultured cardiomyocyte. Therefore, 0.5 mM doxorubicin was chosen to establish the doxorubicin-induced cardiomyocyte necroptosis model in the following experiments.
3.3. Necroptosis inhibitor necrostatin-1 reverses doxorubicininduced cardiotoxicity in cultured cardiomyocytes To further explore whether doxorubicin could induce necroptosis, we pretreated cardiomyocytes with necroptosis specific inhibitor necrostatin-1 (Nec-1) [21]. As shown in Fig. 3A, pretreatment with Nec-1 markedly increased cell viability after doxorubicin treatment. Western blot revealed that Nec-1 substantially decreased expression of RIP1, RIP3 and MLKL, but Nec-1
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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Fig. 2. Doxorubicin reduces cell viability and promotes necroptosis and apoptosis in cardiomyocyte. (A) The cell viability of cardiomyocytes treated with different concentrations of doxorubicin (0, 0.1, 0.5, 1, 2 and 3 mM) for 48 h was measured by MTT assay (n ¼ 3). (B) DNA electrophoresis assay for Con and doxorubicin (0.5, 1, 2 and 3 mM) treated cardiomyocytes. (CeG) RIP1, RIP3, MLKL and cleaved-caspase3 expression, as assayed by Western Blotting, in cardiomyocytes treated with different concentrations of doxorubicin (0, 0.1, 0.5, 1, 2 and 3 mM) for 24 h (n ¼ 3). (*P < 0.05, **P < 0.01, ***P < 0.001 vs the control group).
Fig. 3. Nec-1 and dexrazoxane reverse doxorubicin-induced cardiomyocyte injury. (A) The cell viability of cardiomyocytes as detected by MTT assay after pretreated with Nec-1 (20 mM) for 1 h then treated with doxorubicin (0.5 mM) for 48 h (n ¼ 3). (BeF) Western blots and average data for RIP1, RIP3, MLKL and cleaved-caspase3 in the Con, DOX, DOX þ Nec-1 and Nec-1 groups (n ¼ 3) (G) The cell viability of cardiomyocytes treated with 200 mM dexrazoxane for 3 h prior to 0.5 mM doxorubicin treatment for 24 h was estimated by MTT assay (n ¼ 3). (H) Cell cytotoxicity as evaluated by LDH concentration in the medium after treated with 200 mM dexrazoxane and 0.5 mM doxorubicin (n ¼ 3). (I) DNA electrophoresis assay for Con, DOX, DOX þ DEX, DEX groups (n ¼ 3). (*P < 0.05, **P < 0.01, ***P < 0.001)).
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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increased cleaved caspase-3 in the cardiomyocytes (Fig. 3BeF). It suggested Nec-1 inhibits doxorubicine induced cell necroptosis, but promotes cell apoptosis. Our data demonstrates that Nec-1 could decrease RIP1, RIP3 and MLKL expression; improve cell viability in cardiomyocyte after doxorubicin treatment, which indicates that doxorubicin could cause cardiotoxicity not only by promoting cell apoptosis, but also by cell necroptosis. 3.4. Dexrazoxane increases cell viability and attenuates doxorubicin-induced cardiotoxicity As mentioned above, dexrazoxane is the only FDA approved cardioprotective medicine. To determine whether dexrazoxane exerts cardioprotective effect through its direct action on the cardiomyocytes, we applied dexrazoxane (200 mM) [22] to neonatal mouse ventricular myocytes before doxorubicin treatment. Then we examined cell viability with MTT assay, our outcome illustrated that treatment with dexrazoxane prior to doxorubicin exposure substantially increased the cell viability (Fig. 3G). In addition, we detected LDH release, another index of cellular damage. Doxorubicin induced a remarkable increase in LDH leakage, which was blocked by dexrazoxane treatment (Fig. 3H). DNA electrophoresis assay also proved that dexrazoxane could attenuate doxorubicininduced cell death (Fig. 3I). Our study infers that dexrazoxane could increase cardiomyocyte viability and mitigate cardiotoxicity after doxorubicin treatment.
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3.5. Dexrazoxane alleviates doxorubicin-induced cardiomyocyte necroptosis and apoptosis We next evaluated the impact of dexrazoxane on necroptosis in doxorubicin-induced injury by determining the expression level of RIP1, RIP3 and MLKL. Our data indicated that administration of dexrazoxane resulted in declining the expression levels of RIP1, RIP3, MLKL and cleaved-caspase3 compared to only doxorubicin treatment (Fig. 4AeE). Thus, our results imply that dexrazoxane could inhibit both doxorubicin-induced cardiomyocytes necroptosis and apoptosis.
3.6. Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by p38MAPK/NF-kB pathways To investigate the precise molecular mechanism of dexrazoxane on doxorubicin-induced apoptosis and necroptosis in cardiomyocytes, we analyzed the inflammation signaling pathways including p38MAPK and p65 pathways in response to doxorubicin treatment and dexrazoxane pretreatment. We found that doxorubicin treatment could increase the phosphorylation of p38MAPK and p65, while pretreatment with dexrazoxane could reverse this effect (Fig. 4FeH). Taken together, we can postulate that dexrazoxane may alleviate doxorubicin-induced cardiotoxicity via inhibition of p38MAPK/NF-kB pathway.
Fig. 4. Dexrazoxane alleviates doxorubicin-induced necroptosis in cardiomyocyte. (AeE) The expression of RIP1, RIP3, MLKL and casapse3 in cardiomyocytestreated with or without 200 mM dexrazoxane and 0.5 mM doxorubicin (n ¼ 3). (FeH) Western blotting and average data of phosphorylated-p38MAPK, p38MAPK, phosphorylated-p65, p65 and GAPDH protein abundance in the Con, DOX, DOX þ DEX and DEX groups (n ¼ 3). (*P < 0.05, **P < 0.01, ***P < 0.001).
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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4. Discussion In the present study, our work demonstrated that doxorubicin could reduce the viability of cardiomyocytes in a concentrationdependent manner. Previous studies have shown that oxidative stress injury [4], calcium overload [23], mitochondrial damage [24], apoptosis [25] and autophagy [9,26] are major contributor associated doxorubicin-induced cardiotoxicity. However, the specific mechanism of doxorubicin-induced cardiotoxicity is unclear. Recent studies discovered that Necroptosis plays a prominent role in cardiovascular diseases [27e31]. Unlike necrosis, necroptosis, as one of the programmed cell death, can be reversible. Therefore, we are interested in whether doxorubicin can cause necroptosis in cardiomyocytes. In order to tackle this issue, we firstly used the doxorubicin-induced injury model to simulate necroptosis in cardiomyocytes. We proved that 0.5 mM doxorubicin treatment could lead to necroptosis in cardiomyocytes, which could be reversed by a potent necroptosis inhibitor Nec-1. Dexrazoxane is the only cardiac protective medicine approved by FDA for anthracycline [17]. Many clinical studies have suggested that dexrazoxane could diminish doxorubicin-induced cardiotoxicity in patients [32,33]. Moreover, many animal studies also confirmed that dexrazoxane has a cardioprotective effect on animals treated with doxorubicin [34,35]. Feridooni T et al. [36] and our study found that dexrazoxane could increase the viability of cardiomyocytes and mitigate the cytotoxicity of doxorubicin in vivo and in vitro. So many studies have suggested that dexrazoxane plays a cardioprotective role by reducing inflammation [16] and apoptosis [37]. In our study, we found that dexrazoxane could alleviate doxorubicin-induced both apoptosis and necroptosis in cardiomyocytes. We also found p38MAPK phosphorylation increased significantly in the doxorubicin treated cardiomyocyte in vitro and in vivo [8,38]. Additionally, many previous studies demonstrated that doxorubicin could increase NF-kB activation [39]. Interesting, we found that pretreatment with dexrazoxane could reduce the activation of p38 MAPK and NF-kB after doxorubicin treatment. Thus, we supposed that p38MAPK/NF-kB might be important cell signal pathways in the protective effects of dexrazoxane. In summary, we show here that dexrazoxane exerts a protective effect against doxorubicin-induced cardiotoxicity through attenuating both apoptosis and necroptosis by the inhibition of p38MAPK/ NF-kB pathways in cardiomyocytes. Our findings provide a new mechanism on the cardioprotective effect of dexrazoxane, and suggest that necroptosis may be one of the important signal pathways to treat doxorubicin-induced heart function loss. Funding This study was supported by grants from the National Natural Science Foundation of China (grant no. 81770228, 81470427, 31400995 and 81770858), Beijing Natural Science Foundation (grant no. 7142142, 7154234), Beijing Hospital Nova project (grant no. BJ-2016-045, BJ-2016-034, BJ-2018-138, BJ-2016-032), and NonProfit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018RC310025), Special Subsidy Project of Beijing Municipal Science and Technology Commission on the Application Research of Capital Clinical Characteristics (grant no. Z181100001718138). Author’s contributions Conceived and designed the experiments: XY and TS. Performed the experiments: XY, MY, YR, XH, LD, and TS. Analyzed the data: XY, YR and TS. Contributed reagents/materials/analytical tools: JC, BC,
HG, ML, QW, MY, SS, QQ, XZ, YM, WT, JL. Wrote the paper: XY and TS. All authors read and approved the final manuscript. Ethics approval and consent to participate This study was approved by the Animal Care and Treatment Committee of Beijing Hospital Animal Use and Care Committee and Beijing Normal University Animal Use and Care Committee, and the Guide for Care and Use of Laboratory Animals (NIH Publication # 85-23, revised 1996). Declaration of competing interest The authors declare no conflicts of interest. Acknowledgements Not applicable. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.027 References [1] P. Sharma, S. Kaur, R. Kaur, M. Kaur, S. Kaur, Proteinaceous secretory metabolites of probiotic human commensal Enterococcus hirae 20c, E. Faecium 12a and L12b as antiproliferative agents against cancer cell lines, Front. Microbiol. 9 (2018) 948. [2] Z. Tong, B. Jiang, Y. Wu, Y. Liu, Y. Li, M. Gao, Y. Jiang, Q. Lv, X. Xiao, MiR-21 protected cardiomyocytes against doxorubicin-induced apoptosis by targeting BTG2, Int. J. Mol. Sci. 16 (2015) 14511e14525. [3] Y.L. Chen, X.D. Zhuang, Z.W. Xu, L.H. Lu, H.L. Guo, W.K. Wu, X.X. Liao, Higenamine combined with [6]-Gingerol suppresses doxorubicin-triggered oxidative stress and apoptosis in cardiomyocytes via upregulation of PI3K/ akt pathway, Evid. Based Complement Altern. Med. : eCAM 2013 (2013) 970490. [4] M. Songbo, H. Lang, C. Xinyong, X. Bin, Z. Ping, S. Liang, Oxidative stress injury in doxorubicin-induced cardiotoxicity, Toxicol. Lett. 307 (2019) 41e48. [5] N. Zhao, M.C. Woodle, A.J. Mixson, Advances in delivery systems for doxorubicin, J. Nanomed. Nanotechnol. 9 (2018). [6] Z. Yin, Y. Zhao, H. Li, M. Yan, L. Zhou, C. Chen, D.W. Wang, miR-320a mediates doxorubicin-induced cardiotoxicity by targeting VEGF signal pathway, Aging 8 (2016) 192e207. [7] X. Fang, H. Wang, D. Han, E. Xie, X. Yang, J. Wei, S. Gu, F. Gao, N. Zhu, X. Yin, Q. Cheng, P. Zhang, W. Dai, J. Chen, F. Yang, H.T. Yang, A. Linkermann, W. Gu, J. Min, F. Wang, Ferroptosis as a target for protection against cardiomyopathy, Proc. Natl. Acad. Sci. U. S. A 116 (2019) 2672e2680. [8] Y. Cao, Y. Ruan, T. Shen, X. Huang, M. Li, W. Yu, Y. Zhu, Y. Man, S. Wang, J. Li, Astragalus Polysaccharide Suppresses Doxorubicin-Induced Cardiotoxicity by Regulating the PI3k/Akt and p38MAPK Pathways, Oxidative Medicine and Cellular Longevity, vol. 2014, 2014, p. 674219. [9] Y. Cao, T. Shen, X. Huang, Y. Lin, B. Chen, J. Pang, G. Li, Q. Wang, S. Zohrabian, C. Duan, Y. Ruan, Y. Man, S. Wang, J. Li, Astragalus polysaccharide restores autophagic flux and improves cardiomyocyte function in doxorubicin-induced cardiotoxicity, Oncotarget 8 (2017) 4837e4848. [10] N. Koleini, B.E. Nickel, A.L. Edel, R.R. Fandrich, A. Ravandi, E. Kardami, Oxidized phospholipids in Doxorubicin-induced cardiotoxicity, Chem. Biol. Interact. 303 (2019) 35e39. [11] A. Degterev, Z. Huang, M. Boyce, Y. Li, P. Jagtap, N. Mizushima, G.D. Cuny, T.J. Mitchison, M.A. Moskowitz, J. Yuan, Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury, Nat. Chem. Biol. 1 (2005) 112e119. [12] T. Zhang, Y. Zhang, M. Cui, L. Jin, Y. Wang, F. Lv, Y. Liu, W. Zheng, H. Shang, J. Zhang, M. Zhang, H. Wu, J. Guo, X. Zhang, X. Hu, C.M. Cao, R.P. Xiao, CaMKII is a RIP3 substrate mediating ischemia- and oxidative stress-induced myocardial necroptosis, Nat. Med. 22 (2016) 175e182. [13] A. Roy, S. Sarker, P. Upadhyay, A. Pal, A. Adhikary, K. Jana, M. Ray, Methylglyoxal at metronomic doses sensitizes breast cancer cells to doxorubicin and cisplatin causing synergistic induction of programmed cell death and inhibition of stemness, Biochem. Pharmacol. 156 (2018) 322e339. [14] A.T. Mbaveng, G.T.M. Bitchagno, V. Kuete, P. Tane, T. Efferth, Cytotoxicity of ungeremine towards multi-factorial drug resistant cancer cells and induction of apoptosis, ferroptosis, necroptosis and autophagy, Phytomedicine : Int. J. Phytother. Phytopharm. (2019) 152832. [15] K. McCormack, The cardioprotective effect of dexrazoxane (Cardioxane) is
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Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes Xiaoxue Yu a, b, Yang Ruan c, Xiuqing Huang b, Lin Dou b, Ming Lan b, Ju Cui b, Beidong Chen b, Huan Gong b, Que Wang a, b, Mingjing Yan a, b, Shenghui Sun b, Quan Qiu b, Xiyue Zhang b, Yong Man b, Weiqing Tang b, Jian Li a, b, Tao Shen a, b, * a
Peking University Fifth School of Clinical Medicine, Beijing, 100730, China The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, P.R. China, Beijing, 100730, China c Beijing Tiantan Hospital, Capital Medical University, 100070, Beijing, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 November 2019 Accepted 5 December 2019 Available online xxx
Doxorubicin, as a first line chemotherapeutic agent, its usage is limited owing to cardiotoxicity. Necroptosis is a new form of programmed cell death, and recent investigations indicated that necroptosis is vitally involved in serious cardiac pathological conditions. Dexrazoxane is the only cardiac protective drug approved by FDA for anthracycline. We aimed to explore whether and how dexrazoxane regulates doxorubicin-induced cardiomyocyte necroptosis. First, doxorubicin could cause heart failure and reduce cardiomyocyte viability by promoting cell apoptosis and necroptosis in vivo and in vitro. Second, necroptosis plays an important role in doxorubicin induced cardiomyocyte injury, which could be inhibited by Nec-1. Third, dexrazoxane increased cell viability and protect heart function by decreasing both cardiomyocyte apoptosis and necroptosis after doxorubicin treatment. Forth, dexrazoxane attenuated doxorubicin-induced inflammation and necroptosis by the inhibition of p38MAPK/NF-kB pathways. These results indicated that dexrazoxane ameliorates cardiotoxicity and protects heart function by attenuating both apoptosis and necroptosis in doxorubicin induced cardiomyocyte injury. © 2019 Elsevier Inc. All rights reserved.
Keywords: Dexrazoxane Doxorubicin Necroptosis Apoptosis p38MAPK NF-kB
1. Introduction Cancer is the second leading cause of death worldwide [1]. Doxorubicin (DOX), also known as adriamycin, is an anthracycline antibiotic derived from Streptomyces [2]. Doxorubicin alone or in combination with other chemotherapy has been a common first line therapy for a wide range of cancers, such as lymphoma, leukemia, solid tumor (such as breast cancer and colon cancer) [3e5]. Unfortunately, its clinical usage is hampered by its adverse side effects, most notably a time and dose-dependent cardiotoxicity, which may ultimately result in early or late onset irreversible degenerative cardiomyopathy and congestive heart failure [6]. One of the crucial pathological factors of heart failure is the loss of terminally differentiated cardiomyocytes [7]. Several mechanisms modulating doxorubicin-induced cardiotoxicity have been studied
* Corresponding author. Peking University Fifth School of Clinical Medicine, Beijing, 100730, China. E-mail address: [email protected] (T. Shen).
including apoptosis, dysregulated autophagy, and necrosis [8e10]. Necroptosis is a new form of programmed cell death, which is different from traditional apoptosis and necrosis. Necroptosis is a kind of caspase-independent cell death, mainly mediated by RIPK (receptor interaction protein kinase) and MLKL (mixed lineage kinase domain-like protein) [11]. Necroptosis can trigger inflammation, and plays a significant role in pathological process of various diseases. Recent investigations indicated that necroptosis is vitally involved in serious cardiac pathological conditions, including ischemia-reperfusion injury, myocardial infarction and heart failure [12]. A growing body of evidences confirm that doxorubicin can induce necroptosis in various cancer cell lines [13,14] Previous research also suggests that RIP3 deficiency blocks doxorubicininduced myocardial necroptosis in mice [12]. However, the role of necroptosis in doxorubicin-induced cardiotoxicity remains to be elucidated. Dexrazoxane (DEX) is a water-soluble ring-closed analogue of the iron chelator ethylenediaminetetraacetic acid (EDTA), which belongs to the bisdioxopiperazine class of compounds [15].
https://doi.org/10.1016/j.bbrc.2019.12.027 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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Dexrazoxane is the only FDA approved cardioprotective drug coadministered with anthracycline for preventing anthracyclineinduced cardiac damage [6]. Current studies show that dexrazoxane can reduce oxidative stress by means of chelating free iron and prevent anthracycline from binding to the topisomerase 2b complex [16]. The precise cardioprotective mechanism of dexrazoxane is not fully understood, and it is still unclear whether and how dexrazoxane regulates doxorubicin-induced injury in cardiomyocyte. In the present study, we established a doxorubicin-induced mouse heart failure model in vivo and cardiomyocytes injury model to determine the protective mechanism of dexrazoxane on cardiomyocytes, which may offer new evidence for the cardioprotective mechanisms of dexrazoxane.
deoxyuridine (Sigma-Aldrich, USA) and incubated at 37 C in a humidified atmosphere with 5% CO2 and 95% air (v/v). 2.5. Preparation of drugs Doxorubicin hydrochloride was purchased from the Cell signaling Technology (Beverly, MA, US). The doxorubicin hydrochloride was dissolved in deionized water to 5 mM and then diluted with DMEM culture medium containing 1% FBS at different concentrations. Dexrazoxane (DEX) and Nec-1 were purchased from the Sigma-Aldrich, USA, DEX and Nec-1 were separately dissolved in DMSO to 60 mM and 10 mM, then diluted them to different concentrations with DMEM culture medium containing 1% FBS. 2.6. Cell viability and lactate dehydrogenase (LDH) leakage assays
2. Materials and methods 2.1. Animals Ten-week-old male C57BL/6 mice were provided by SPF (Beijing) Biotechnology Co. The mice (n ¼ 40) were randomly divided into 4 groups. Control (Con) mice were injected 0.1 ml of 0.167 mol/ L sodium lactate solution and saline as placebo intraperitoneally. Doxorubicin-treated mice (DOX) were injected with doxorubicin hydrochloride dissolved in normal saline (10 mg/kg i.p.) three times at day 1, 3 and 5, and 0.1 ml of 0.167 mol/L sodium lactate solution 3 h before DOX treatment. Doxorubicin plus Dexrazoxane treatment mice (DOX þ DEX) were pretreated with 0.1 ml DEX solution (200 mg/kg/day, dissolved in 0.167 mol/L sodium lactate solution) 3 h before 10 mg/kg DOX treatment for three times at day 1, 3 and 5 intraperitoneally. Dexrazoxane-treated mice (DEX) were injected 0.1 ml DEX solution. The dosage of DOX and DEX was modified according to previous studies [17]. All of the mice in the 4 groups were euthanized 7 days after the initial injection of doxorubicin. All animal experiments conformed to the protocols approved by Beijing Hospital Animal Use and Care Committee, and the Guide for Care and Use of Laboratory Animals (NIH Publication # 85-23, revised 1996). 2.2. Echocardiography Mice were lightly anesthetized with 1e1.5% isoflurane in oxygen until the heart rate stabilized to 400 to 500 beats per minute. Echocardiography was performed using Vevo 770 and Vevo 2100 (VisualSonics) instruments. Fraction shortening (FS), ejection fraction (EF), left ventricular internal diameter (LVID) during systole, LVID during diastole, end-systolic volume, and end-diastolic volume were calculated with Vevo Analysis software (version 2.2.3) as previously described [8]. After echocardiography measurements, mice were euthanatized by cervical dislocation, and the hearts were collected for further analyses. 2.3. DNA electrophoresis assay DNA electrophoresis assay was performed as previously described [18]. 2.4. Neonatal mouse ventricular myocyte isolation and culture Neonatal mouse ventricular myocytes (NMVMs) were isolated from 1-3-day-old C57BL/6J mice by digestion with the combination of trypsin and collagenase type II [19]. Cardiomyocytes were plated at a density of 6.6 104 cells/cm2 in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA) in the presence of 0.1 mM 5-bromo-2-
Cells (5 103 cells/well) were seeded in 96-well plates, the cells were divided into four groups: Con groups were treated with DMSO alone as control, DOX group were treated with 0.5 mM of doxorubicin hydrochloride, DOX þ DEX group were pretreated with 200 mM dexrazoxane 4 h before 0.5 mM of doxorubicin hydrochloride treatment, and DEX group were treated with 200 mM dexrazoxane. Then the cells viability was determined with MTT assay kit (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-aldrich, US) according to the manufacturer’s protocol. The LDH concentration was detected using an LDH assay kit according to the manufacturer’s protocol (Solarbio, Beijing, China). The absorbance was obtained by a spectrophotometer. All assays were performed at least three times. 2.7. Western blot analysis Western blotting was performed as previously described [20]. 2.8. Statistical analysis Values are presented as the means ± SEM of at least three experiments. Data were analyzed using GraphPad Prism 6.0 statistical software (GraphPad Software, CA, USA). The differences between two groups were analyzed using Student’s t-test, and multiple comparisons were analyzed using one-way ANOVA with Bonferroni’s correction. For all tests, probability value less than 0.05 was defined as statistically significant. 3. Results 3.1. Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity and protects heart function in vivo To determine whether dexrazoxane could protect heart function after doxorubicin treatment in vivo, we generated a heart failure model using doxorubicin-treated male C57BL/6J mice. Lower body weight and higher heart/body weight ration were observed in control mice than doxorubicin-treated mice, which could be improved by dexrazoxane. (Fig. 1A and B). Meanwhile, we measured heart function by Echo analysis. Compared with control mice, doxorubicin-treated mice exhibited decreased heart function as measured by the ejection fraction (EF) % and fraction shortening index (FS) % upon echocardiogram. However, pretreatment with DEX significantly preserved heart function in vivo (Fig. 1CeE). Moreover, Western blot analysis revealed that DEX suppressed doxorubicin-induced RIP1, RIP3 MLKL and cleaved-caspase 3 (active form of caspase-3) expression in mouse heart (Fig. 1FeJ). These results suggested that doxorubicin could cause cardiotoxicity and heart function loss by promoting both apoptosis
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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Fig. 1. Dexrazoxane ameliorates doxorubicin-caused necroptosis and apoptosis in vivo. (AeB) Body weight and ratio of heart weight to body weight in Control mouse (Con), doxorubicin treated mouse (DOX), doxorubicin and dexrazoxane pretreated mouse (DOX þ DEX), dexrazoxane treated mouse (DEX). (CeE) Echocardiography of Con, DOX, DOX þ DEX, DEX mouse heart function 7 days after fist treatment as shown by fractional shortening % (FS %) and ejection fraction % (EF %) (n ¼ 6, *p < 0.05, **p < 0.01). (FeJ) Western blotting and average data for RIP1, RIP3, MLKL and cleaved caspase 3 in Con, DOX, DOX þ DEX, DEX groups 7 days after fist treatment (n ¼ 5, *p < 0.05, **p < 0.01).
and necroptosis. Dexrazoxane treatment remarkably alleviated the cardiotoxicity and the heart function loss in vivo.
3.2. Doxorubicin induces cardiotoxicity by promoting both apoptosis and necroptosis in cardiomyocytes Cell viability assay was used to determine the viability of neonatal mouse ventricular myocytes (NMVMs) after doxorubicin treatment. We found that the cell activity decreased with the increase of doxorubicin concentration in a concentration-dependent manner (Fig. 2A). Moreover, in order to reveal the effects of doxorubicin treatment on different kinds of cell death type, we used DNA electrophoresis assay to analyze cell death type. DNA electrophoresis shows that both DNA smear and DNA laddering appeared at the 0.5 mM of DOX treatment, suggesting both necrosis and apoptosis existed in the doxorubicin treated cardiomyocytes. When increased the doxorubicin concentration to 1e3 mM, there is only DNA smear in the cardiomyocyte, which suggests that high concentration of DOX leads to classic necrosis (Fig. 2B). Further, we analyzed cell apoptosis and necroptosis in doxorubicin treated cardiomyocytes. Necroptosis related proteins such as RIP1, RIP3,
MLKL and apoptosis related cleaved caspase-3 were detected by Western blotting. Our data manifested that both RIP1, RIP3 and MLKL were significantly upregulated when exposed to 0.5 mM doxorubicin treatment compared with the control. While cleaved caspase-3 (active form of caspase-3) increased obviously at the doxorubicin concentration of 1 mMe3 mM (Fig. 2CeG). All these results suggest that low concentration of doxorubicin (0.5 mM) could induce cardiotoxicity by increasing both cell apoptosis and cell necrosis in cultured cardiomyocyte. Therefore, 0.5 mM doxorubicin was chosen to establish the doxorubicin-induced cardiomyocyte necroptosis model in the following experiments.
3.3. Necroptosis inhibitor necrostatin-1 reverses doxorubicininduced cardiotoxicity in cultured cardiomyocytes To further explore whether doxorubicin could induce necroptosis, we pretreated cardiomyocytes with necroptosis specific inhibitor necrostatin-1 (Nec-1) [21]. As shown in Fig. 3A, pretreatment with Nec-1 markedly increased cell viability after doxorubicin treatment. Western blot revealed that Nec-1 substantially decreased expression of RIP1, RIP3 and MLKL, but Nec-1
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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Fig. 2. Doxorubicin reduces cell viability and promotes necroptosis and apoptosis in cardiomyocyte. (A) The cell viability of cardiomyocytes treated with different concentrations of doxorubicin (0, 0.1, 0.5, 1, 2 and 3 mM) for 48 h was measured by MTT assay (n ¼ 3). (B) DNA electrophoresis assay for Con and doxorubicin (0.5, 1, 2 and 3 mM) treated cardiomyocytes. (CeG) RIP1, RIP3, MLKL and cleaved-caspase3 expression, as assayed by Western Blotting, in cardiomyocytes treated with different concentrations of doxorubicin (0, 0.1, 0.5, 1, 2 and 3 mM) for 24 h (n ¼ 3). (*P < 0.05, **P < 0.01, ***P < 0.001 vs the control group).
Fig. 3. Nec-1 and dexrazoxane reverse doxorubicin-induced cardiomyocyte injury. (A) The cell viability of cardiomyocytes as detected by MTT assay after pretreated with Nec-1 (20 mM) for 1 h then treated with doxorubicin (0.5 mM) for 48 h (n ¼ 3). (BeF) Western blots and average data for RIP1, RIP3, MLKL and cleaved-caspase3 in the Con, DOX, DOX þ Nec-1 and Nec-1 groups (n ¼ 3) (G) The cell viability of cardiomyocytes treated with 200 mM dexrazoxane for 3 h prior to 0.5 mM doxorubicin treatment for 24 h was estimated by MTT assay (n ¼ 3). (H) Cell cytotoxicity as evaluated by LDH concentration in the medium after treated with 200 mM dexrazoxane and 0.5 mM doxorubicin (n ¼ 3). (I) DNA electrophoresis assay for Con, DOX, DOX þ DEX, DEX groups (n ¼ 3). (*P < 0.05, **P < 0.01, ***P < 0.001)).
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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increased cleaved caspase-3 in the cardiomyocytes (Fig. 3BeF). It suggested Nec-1 inhibits doxorubicine induced cell necroptosis, but promotes cell apoptosis. Our data demonstrates that Nec-1 could decrease RIP1, RIP3 and MLKL expression; improve cell viability in cardiomyocyte after doxorubicin treatment, which indicates that doxorubicin could cause cardiotoxicity not only by promoting cell apoptosis, but also by cell necroptosis. 3.4. Dexrazoxane increases cell viability and attenuates doxorubicin-induced cardiotoxicity As mentioned above, dexrazoxane is the only FDA approved cardioprotective medicine. To determine whether dexrazoxane exerts cardioprotective effect through its direct action on the cardiomyocytes, we applied dexrazoxane (200 mM) [22] to neonatal mouse ventricular myocytes before doxorubicin treatment. Then we examined cell viability with MTT assay, our outcome illustrated that treatment with dexrazoxane prior to doxorubicin exposure substantially increased the cell viability (Fig. 3G). In addition, we detected LDH release, another index of cellular damage. Doxorubicin induced a remarkable increase in LDH leakage, which was blocked by dexrazoxane treatment (Fig. 3H). DNA electrophoresis assay also proved that dexrazoxane could attenuate doxorubicininduced cell death (Fig. 3I). Our study infers that dexrazoxane could increase cardiomyocyte viability and mitigate cardiotoxicity after doxorubicin treatment.
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3.5. Dexrazoxane alleviates doxorubicin-induced cardiomyocyte necroptosis and apoptosis We next evaluated the impact of dexrazoxane on necroptosis in doxorubicin-induced injury by determining the expression level of RIP1, RIP3 and MLKL. Our data indicated that administration of dexrazoxane resulted in declining the expression levels of RIP1, RIP3, MLKL and cleaved-caspase3 compared to only doxorubicin treatment (Fig. 4AeE). Thus, our results imply that dexrazoxane could inhibit both doxorubicin-induced cardiomyocytes necroptosis and apoptosis.
3.6. Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by p38MAPK/NF-kB pathways To investigate the precise molecular mechanism of dexrazoxane on doxorubicin-induced apoptosis and necroptosis in cardiomyocytes, we analyzed the inflammation signaling pathways including p38MAPK and p65 pathways in response to doxorubicin treatment and dexrazoxane pretreatment. We found that doxorubicin treatment could increase the phosphorylation of p38MAPK and p65, while pretreatment with dexrazoxane could reverse this effect (Fig. 4FeH). Taken together, we can postulate that dexrazoxane may alleviate doxorubicin-induced cardiotoxicity via inhibition of p38MAPK/NF-kB pathway.
Fig. 4. Dexrazoxane alleviates doxorubicin-induced necroptosis in cardiomyocyte. (AeE) The expression of RIP1, RIP3, MLKL and casapse3 in cardiomyocytestreated with or without 200 mM dexrazoxane and 0.5 mM doxorubicin (n ¼ 3). (FeH) Western blotting and average data of phosphorylated-p38MAPK, p38MAPK, phosphorylated-p65, p65 and GAPDH protein abundance in the Con, DOX, DOX þ DEX and DEX groups (n ¼ 3). (*P < 0.05, **P < 0.01, ***P < 0.001).
Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027
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4. Discussion In the present study, our work demonstrated that doxorubicin could reduce the viability of cardiomyocytes in a concentrationdependent manner. Previous studies have shown that oxidative stress injury [4], calcium overload [23], mitochondrial damage [24], apoptosis [25] and autophagy [9,26] are major contributor associated doxorubicin-induced cardiotoxicity. However, the specific mechanism of doxorubicin-induced cardiotoxicity is unclear. Recent studies discovered that Necroptosis plays a prominent role in cardiovascular diseases [27e31]. Unlike necrosis, necroptosis, as one of the programmed cell death, can be reversible. Therefore, we are interested in whether doxorubicin can cause necroptosis in cardiomyocytes. In order to tackle this issue, we firstly used the doxorubicin-induced injury model to simulate necroptosis in cardiomyocytes. We proved that 0.5 mM doxorubicin treatment could lead to necroptosis in cardiomyocytes, which could be reversed by a potent necroptosis inhibitor Nec-1. Dexrazoxane is the only cardiac protective medicine approved by FDA for anthracycline [17]. Many clinical studies have suggested that dexrazoxane could diminish doxorubicin-induced cardiotoxicity in patients [32,33]. Moreover, many animal studies also confirmed that dexrazoxane has a cardioprotective effect on animals treated with doxorubicin [34,35]. Feridooni T et al. [36] and our study found that dexrazoxane could increase the viability of cardiomyocytes and mitigate the cytotoxicity of doxorubicin in vivo and in vitro. So many studies have suggested that dexrazoxane plays a cardioprotective role by reducing inflammation [16] and apoptosis [37]. In our study, we found that dexrazoxane could alleviate doxorubicin-induced both apoptosis and necroptosis in cardiomyocytes. We also found p38MAPK phosphorylation increased significantly in the doxorubicin treated cardiomyocyte in vitro and in vivo [8,38]. Additionally, many previous studies demonstrated that doxorubicin could increase NF-kB activation [39]. Interesting, we found that pretreatment with dexrazoxane could reduce the activation of p38 MAPK and NF-kB after doxorubicin treatment. Thus, we supposed that p38MAPK/NF-kB might be important cell signal pathways in the protective effects of dexrazoxane. In summary, we show here that dexrazoxane exerts a protective effect against doxorubicin-induced cardiotoxicity through attenuating both apoptosis and necroptosis by the inhibition of p38MAPK/ NF-kB pathways in cardiomyocytes. Our findings provide a new mechanism on the cardioprotective effect of dexrazoxane, and suggest that necroptosis may be one of the important signal pathways to treat doxorubicin-induced heart function loss. Funding This study was supported by grants from the National Natural Science Foundation of China (grant no. 81770228, 81470427, 31400995 and 81770858), Beijing Natural Science Foundation (grant no. 7142142, 7154234), Beijing Hospital Nova project (grant no. BJ-2016-045, BJ-2016-034, BJ-2018-138, BJ-2016-032), and NonProfit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018RC310025), Special Subsidy Project of Beijing Municipal Science and Technology Commission on the Application Research of Capital Clinical Characteristics (grant no. Z181100001718138). Author’s contributions Conceived and designed the experiments: XY and TS. Performed the experiments: XY, MY, YR, XH, LD, and TS. Analyzed the data: XY, YR and TS. Contributed reagents/materials/analytical tools: JC, BC,
HG, ML, QW, MY, SS, QQ, XZ, YM, WT, JL. Wrote the paper: XY and TS. All authors read and approved the final manuscript. Ethics approval and consent to participate This study was approved by the Animal Care and Treatment Committee of Beijing Hospital Animal Use and Care Committee and Beijing Normal University Animal Use and Care Committee, and the Guide for Care and Use of Laboratory Animals (NIH Publication # 85-23, revised 1996). Declaration of competing interest The authors declare no conflicts of interest. Acknowledgements Not applicable. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.027 References [1] P. Sharma, S. Kaur, R. Kaur, M. Kaur, S. Kaur, Proteinaceous secretory metabolites of probiotic human commensal Enterococcus hirae 20c, E. Faecium 12a and L12b as antiproliferative agents against cancer cell lines, Front. Microbiol. 9 (2018) 948. [2] Z. Tong, B. Jiang, Y. Wu, Y. Liu, Y. Li, M. Gao, Y. Jiang, Q. Lv, X. Xiao, MiR-21 protected cardiomyocytes against doxorubicin-induced apoptosis by targeting BTG2, Int. J. Mol. Sci. 16 (2015) 14511e14525. [3] Y.L. Chen, X.D. Zhuang, Z.W. Xu, L.H. Lu, H.L. Guo, W.K. Wu, X.X. Liao, Higenamine combined with [6]-Gingerol suppresses doxorubicin-triggered oxidative stress and apoptosis in cardiomyocytes via upregulation of PI3K/ akt pathway, Evid. Based Complement Altern. Med. : eCAM 2013 (2013) 970490. [4] M. Songbo, H. Lang, C. Xinyong, X. Bin, Z. Ping, S. Liang, Oxidative stress injury in doxorubicin-induced cardiotoxicity, Toxicol. Lett. 307 (2019) 41e48. [5] N. Zhao, M.C. Woodle, A.J. Mixson, Advances in delivery systems for doxorubicin, J. Nanomed. Nanotechnol. 9 (2018). [6] Z. Yin, Y. Zhao, H. Li, M. Yan, L. Zhou, C. Chen, D.W. Wang, miR-320a mediates doxorubicin-induced cardiotoxicity by targeting VEGF signal pathway, Aging 8 (2016) 192e207. [7] X. Fang, H. Wang, D. Han, E. Xie, X. Yang, J. Wei, S. Gu, F. Gao, N. Zhu, X. Yin, Q. Cheng, P. Zhang, W. Dai, J. Chen, F. Yang, H.T. Yang, A. Linkermann, W. Gu, J. Min, F. Wang, Ferroptosis as a target for protection against cardiomyopathy, Proc. Natl. Acad. Sci. U. S. A 116 (2019) 2672e2680. [8] Y. Cao, Y. Ruan, T. Shen, X. Huang, M. Li, W. Yu, Y. Zhu, Y. Man, S. Wang, J. Li, Astragalus Polysaccharide Suppresses Doxorubicin-Induced Cardiotoxicity by Regulating the PI3k/Akt and p38MAPK Pathways, Oxidative Medicine and Cellular Longevity, vol. 2014, 2014, p. 674219. [9] Y. Cao, T. Shen, X. Huang, Y. Lin, B. Chen, J. Pang, G. Li, Q. Wang, S. Zohrabian, C. Duan, Y. Ruan, Y. Man, S. Wang, J. Li, Astragalus polysaccharide restores autophagic flux and improves cardiomyocyte function in doxorubicin-induced cardiotoxicity, Oncotarget 8 (2017) 4837e4848. [10] N. Koleini, B.E. Nickel, A.L. Edel, R.R. Fandrich, A. Ravandi, E. Kardami, Oxidized phospholipids in Doxorubicin-induced cardiotoxicity, Chem. Biol. Interact. 303 (2019) 35e39. [11] A. Degterev, Z. Huang, M. Boyce, Y. Li, P. Jagtap, N. Mizushima, G.D. Cuny, T.J. Mitchison, M.A. Moskowitz, J. Yuan, Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury, Nat. Chem. Biol. 1 (2005) 112e119. [12] T. Zhang, Y. Zhang, M. Cui, L. Jin, Y. Wang, F. Lv, Y. Liu, W. Zheng, H. Shang, J. Zhang, M. Zhang, H. Wu, J. Guo, X. Zhang, X. Hu, C.M. Cao, R.P. Xiao, CaMKII is a RIP3 substrate mediating ischemia- and oxidative stress-induced myocardial necroptosis, Nat. Med. 22 (2016) 175e182. [13] A. Roy, S. Sarker, P. Upadhyay, A. Pal, A. Adhikary, K. Jana, M. Ray, Methylglyoxal at metronomic doses sensitizes breast cancer cells to doxorubicin and cisplatin causing synergistic induction of programmed cell death and inhibition of stemness, Biochem. Pharmacol. 156 (2018) 322e339. [14] A.T. Mbaveng, G.T.M. Bitchagno, V. Kuete, P. Tane, T. Efferth, Cytotoxicity of ungeremine towards multi-factorial drug resistant cancer cells and induction of apoptosis, ferroptosis, necroptosis and autophagy, Phytomedicine : Int. J. Phytother. Phytopharm. (2019) 152832. [15] K. McCormack, The cardioprotective effect of dexrazoxane (Cardioxane) is
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Please cite this article as: X. Yu et al., Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.027