Fucoxanthin attenuates doxorubicin-induced cardiotoxicity via anti-oxidant and anti-apoptotic mechanisms associated with p38, JNK and p53 pathways

Journal of Functional Foods 62 (2019) 103542

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Fucoxanthin attenuates doxorubicin-induced cardiotoxicity via anti-oxidant and anti-apoptotic mechanisms associated with p38, JNK and p53 pathways Yu-Qin Zhao , Lun Zhang, Guo-Xu Zhao, Yin Chen, Kun-Lai Sun, Bin Wang ⁎

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Zhejiang Provincial Engineering Technology Research Center of Marine Biomedical Products, School of Food and Pharmacy, Zhejiang Ocean University, 1st Haidanan Road, Changzhi Island, Lincheng, Zhoushan, Zhejiang 316022, PR China

ARTICLE INFO

ABSTRACT

Keywords: Fucoxanthin Doxorubicin Reactive oxygen species Mitogen-activated protein kinase P53 Apoptosis

Doxorubicin (DOX) is a commonly used anthracycline in treatments of leukemia, lymphoma and breast cancer in humans and has been limited by its cardiotoxicity, which is manifested congestive heart failure in the worst condition. The present study showed that the marine carotenoid of fucoxanthin could exert cardioprotective effect against the DOX-induced injury in ICR mice. And then, the protective effects and mechanisms of fucoxanthin on DOX-induced injury of neonatal rat cardiomyocytes were investigated. The results demonstrated that fucoxanthin significantly reduced mice toxic death triggered by DOX. The protective effects were also showed by the suppression of apoptotic cell death in cardiomyocytes. Further study revealed that fucoxanthin-produced suppression of MAPK activated by DOX was involved in the cardioprotection. The findings confirmed the cardioprotective effect of fucoxanthin against DOX-induced cardiotoxicity by protecting myocardial cells from lipid peroxidation and apoptosis, which would likely result in an increased therapeutic window of DOX in cancer therapy.

1. Introduction Doxorubicin, which was the first anthracycline isolated, is widely used in the treatment of a variety of tumor types, including leukemia (Bhowmik et al., 2017), multiple myeloma (Mittenberg et al., 2018), neuroblastoma (Sagnella et al., 2018), sarcoma (Jamieson et al., 2017), lymphoma and so forth (Bonadonna, Monfardini, De Lena, FossatiBellani, & Beretta, 1970; Bonadonna, Monfardini, De, & Fossati-Bellani, 1969). However, a serious cardiac adverse effects limited its clinical use (Cristina et al., 2009; Praga et al., 1979). Increasing studies indicated that fatal congestive heart failure (CHF), the most severe form of DOXinduced myocardial toxicity, may occur either during the DOX therapy or a period of time after the therapy, and the chronic cardiotoxicity is dose-dependent (Theodoulou & Hudis, 2004). Retrospective analysis indicated that an estimated cumulative 26% of patients would experience DOX-induced CHF at a cumulative dose of 550 mg/m2, and the mortality rate would nearly reach up to 30–50% (Theodoulou & Hudis, 2004). To mitigate DOX-related cardiotoxicity, several strategies have been considered: dosage optimization, use of analogues or combined therapy like antioxidants (Delemasure, Vergely, Zeller, Cottin, & Rochette, 2006). Among them, increasing attention has been paid to the development of cardioprotective agents. In the last 50 years, to accomplish more successful prevention or ⁎

intervention of DOX cardiotoxicity, a number of efforts have been exerted to find the effective strategies. Several cardioprotective agents including phenylbutyrate (Chotiros et al., 2007), amifostine (Bolaman et al., 2005), glutathione (GSH) (ElSwefy, Mohamed, & Hagar, 2000), and some natural compounds, such as bergamot polyphenols (Carresi et al., 2018), have been studied. Currently, the most effective compound, dexrazoxane, discovered by Kurt Hellmann in 1972, is used as a cardioprotectant in clinical (Blum, 1997; Wouters, Kremer, Miller, Herman, & Lipshultz, 2010). The mechanistic studies showed that dexrazoxane could potentially displace cellular iron and prevents cardiotoxicity associated with DOX (Hasinoff & Herman, 2007; Hasinoff, Hellmann, Herman, & Ferrans, 2010). However, the clinical trials demonstrated that dexrazoxane might interfere with the anticancer activity of DOX and lead to the higher underlying risk for Myelodysplastic syndrome and secondary acute myeloid leukemia (AML) in pediatric patients receiving dexrazoxane (Salzer et al., 2010; Tebbi et al., 2007). These studies suggest that it is not sufficient in finding safer and more effective cardioprotective agents. Therefore, development of new cardioprotective agents which can attenuate the DOX-induced cardiotoxicity without reducing the anticancer potency remains the research hotspot. Fucoxanthin (FUC) (Fig. 1A) is an allenic carotenoid and isolated from marine edible brown seaweeds. Recent research has reported that

Corresponding authors. E-mail addresses: [email protected] (Y.-Q. Zhao), [email protected] (B. Wang).

https://doi.org/10.1016/j.jff.2019.103542 Received 14 May 2019; Received in revised form 21 August 2019; Accepted 26 August 2019 Available online 06 September 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Fucoxanthin reduced doxorubicin-induced cardiotoxicities in vivo. (A) The structure of fucoxanthin. (B) Survival rates of ICR mice receiving DOX (20 mg/kg, ip) with or without FUC as described in Materials and Methods (n = 10). (C) The mice serum concentrations of AST, LDH and CKMB were analyzed as described above. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01 compared to control group. #P < 0.05, ##P < 0.01 compared to DOX alone group.

from the Institute of Cell Biology in Shanghai. The cells were cultured in RPMI-1640 (GIBCO, Grand Island, USA), supplemented with penicillin (100 U/ml), streptomycin (100 U/ml) and 10% FBS (Hyclone, Logan, UT, USA). All the cells were cultured at 37 °C with 5% CO2 in a humidified atmosphere.

FUC has remarkable biological properties (Viera, Perez-Galvez, & Roca, 2018), such as antiobesity (Zhang et al., 2015), antitumor (Mei et al., 2017), antidiabetes (Lin, Tsou, Chen, Lu, & Hwang, 2017), antioxidant (Maeda, Fukuda, Izumi, & Saga, 2018), anti-inflammatory (Zhang et al., 2015), and hepatoprotective activities (Corona et al., 2016), as well as cerebrovascular and cardiovascular protective effects (Seo et al., 2016). In this study, the cardioprotective activity of FUC was investigated in DOX-treated primary myocardial cells and mice models. And further study indicated that FUC exhibited a synergetic effect with DOX in the anticancer activity. Taken together, these findings represent here was undertaken to evaluate the dual mode of FUC on the cardioprotective effect and anticancer activity, which show the promising potential of FUC as a cardioprotectant in the clinical application of DOX.

2.3. The ICR mice model of cardiac toxicity of doxorubicin and the measurement of AST, LDH and CKMB leakage DOX and FUC were freshly dissolved in sterile double distilled water, immediately before use. The animals were injected intraperitoneally either with 0.01 ml/g body weight DOX or FUC. The ICR mice were divided into vehicle-treated group (control, n = 10) which received a constant volume of dH2O, and 20 mg/kg DOX-treated group (n = 10), 20 mg/kg DOX and 125 mg/kg FUC-treated group (n = 10), 20 mg/kg DOX and 250 mg/kg FUC-treated group (n = 10), 20 mg/kg DOX and 500 mg/kg FUC-treated group (n = 10) randomly. The ICR mice were treated with FUC (ig) for 4 days, then, DOX were treated (ip) on the fourth day to make the acute cardiac injury and the mice were tested for another 6 days. The death rate of the mice was measured every day. Serum cardiac enzyme activity of AST, LDH and CKMB were detected in the sixth day after drawing blood through carotid artery and determined by full-automatic biochemical detect machine (Cobas c311, Roche Diagnostics GmbH, Germany) using specific detective kits.

2. Materials and methods 2.1. Chemicals and reagents Doxorubicin was purchased from Zhejiang Haizheng Pharmaceutical Co Ltd (Zhejiang Province, China). Fucoxanthin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for PARP, caspase 3, Bcl-2, Bax, phosphorylated (P)-ERK, phosphorylated (P)-p38, phosphorylated (P)-JNK, β-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and phosphorylated (p)-p53 from Cell Signal Technology Inc. (Beverly, Massachusetts, USA). The HRP-labeled secondary anti-mouse, anti-goat and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The western blot detection reagent ECL was purchased from Pierce Biotechnology (Rockford, IL, USA).

2.4. Neonatal rat cardiomyocytes preparation Neonatal rat cardiomyocytes were prepared from 2 to 3-day-old neonatal Sprague-Dawley rats. Rats were euthanized by cervical dislocation and the cardiac apexes were retained only. The obtained tissue was kept in ice-cold D-Hank’s balanced salt solution without Ca2+ and Mg2+ ions, washed three times with the same solution and minced into small fragments. Then cardiac cells were dissociated with trypsin

2.2. Cancer cell culture Human leukemia cancer cell line U937 and K562 were purchased 2

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(0.06%, w/v) for three or four times and obtained by centrifuging the combined digestion. To exclude non-muscle cells, the isolated cells were first plated in tissue culture dishes at 37 °C for 4 h, and non-adherent cells were collected then incubated with bromodeoxyuridine (BrdU, 0.1 mmol/L) in the medium for three days to inhibit fibroblast growth. The neonatal rat cardiomyocytes were routine cultured in media DMEM, changed every two or three days, supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 U/ml). After additional 72 h incubation, the monolayer cells were used in the experiments.

JC-1 kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. Cells were rinsed with PBS and incubated with JC-1 staining solution at 37 °C for 20 min. The fluorescent signal was measured at 525 nm (green) and 590 nm (red) wavelengths. Ratio of red to green fluorescence was calculated and analyzed. 2.10. Measurement of intracellular GSH The neonatal rat cardiomyocytes treated with different drugs for 24 h were harvested, lysed and the supernatants were collected to detect the content of GSH using an assay kits (Nanjing Jiancheng, China) according to the manufacturer’s protocol.

2.5. Cytotoxicity assay Cells were seeded into 96-well plates with an appropriate density. After pretreatment with varying concentrations of FUC for 24 h, cells were exposed to DOX for another 24 h and the proliferation inhibition rate were determined using MTT assay. 5 mg/ml MTT solution was added (20.0 μL/well) and the plates were incubated for 4 h at 37 °C. The purple formazan crystals produced in cells were dissolved in 100 μL DMSO and read on an automated microplate spectrophotometer (Thermo Multiskan Spectrum, Thermo Electron Corporation, Vantaa, Finland) at 570 nm.

2.11. DNA gel electrophoresis assay The neonatal rat cardiomyocytes (1 × 106) were treated with different drugs for 48 h, DNA fragmentation was extracted as previous report (Steinfelder, Quentin, & Ritz, 2000). Briefly, harvested cells were lysed by equal volumes of 1.2% SDS. By adding 7/10 vol of the precipitation solution (3 mol/L CsCl, 1 mol/L potassium acetate, 0.67 mol/ L acetic acid) and spinning for 15 min at 14,000 rpm, DNA fragmentation was kept in the supernatant, which was then absorbed by a miniprep spin column. Finally, DNA was eluted with 50 ml TE buffer (pH, 8.0) and electrophoresed in parallel on the same 2% agarose gel in 1 × TAE buffer with 0.5 mg/mL ethidium bromide staining to facilitate visualization by fluorescence under UV light. Molecular weight standards (Trans2K Plus II DNA Marker, Transgen, China) were run on the left-hand lane of the same gel. Images were captured using Bio-Rad GD2000 (Bio-Rad, CA, USA).

2.6. DAPI staining assay Neonatal rat cardiomyocytes were cultured in 24-well plates and pretreated with or without FUC (50 μM) for 24 h followed by another incubation with DOX (2 μM) for 48 h. Washed the cells twice with PBS and then incubated with 4′,6-diamidino-2-phenylindole (DAPI) which was diluted with 0.1% Triton X-100 for 5 min. Photographed the changes of nuclei with fluorescence microscope (DMI 4000B, Leica, Germany).

2.12. Western blot analysis The protein samples of the neonatal rat cardiomyocytes were extracted in lysate buffer and the total protein concentration of whole cell lysates was determined using the Bradford method (Bio-Rad, CA, USA). 40.0–60.0 μg of total protein was loaded per lane and fractionated on 10–15% tris–glycine precast gels, transferred to PVDF membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dry milk in 0.01 M Tris Buffered Saline with 0.1% Tween-20 (TBST) for 1 h. Subsequently, the membrane was incubated with primary antibodies (1:500) directed against target proteins overnight at 4 °C and HRP-labeled secondary antibodies diluted at 1:5000 in TBST for 1 h. Proteins were visualized using ECL.

2.7. DPPH radical scavenging assay The DPPH radical scavenging capacity was detected as follows (Wang, Li, Chi, Zhang, & Luo, 2012). The different concentration of FUC and vitamin C was prepared and a volume of 2 μL of each sample was added to an ethanolic solution of DPPH (25 μg/mL) to a final volume of 200 μL for 30 min in 96-well plate. The decrease in absorbance at 517 nm was determined with an automated microplate spectrophotometer (Thermo Multiskan Spectrum, Thermo Electron Corporation, Vantaa, Finland). The degree of DPPH radical scavenging activity was calculated as follows: inhibition rate (%) = (A517blank − A517sample)/A517blank × 100%.

2.13. Statistical analysis

2.8. Measurement of intracellular ROS

All values were expressed as mean ± SD. The non-parametric U Mann Whitney test was employed to analyze the data. For each analysis, three independent experiments were conducted to obtain the data. * and # indicates the values are significantly different than the control.

The neonatal rat cardiomyocytes were cultured in 24-well plates (5 × 104/ml). After the drug treatment for 3 h, the level of intracellular ROS was measured using the oxidation sensitive fluorescent dye Carboxy-DCFDA. An increase in green fluorescence intensity is used to quantify the generation of intracellular ROS. Carboxy-DCFDA was added at a final concentration of 15 μM to the cell suspension, dissociated by EDTA, the neonatal rat cardiomyocytes were incubated at 37 °C for 30 min, washed with PBS, and measured immediately by fluorescence spectrometer (Becton Dickinson, CA, USA) using an argon laser at 488 nm and a 535 nm bandpass filter. The fold change of ROS level was calculated as fluorescence per μg protein compared with untreated cells.

3. Results 3.1. Fucoxanthin protects against doxorubicin-induced cardiotoxicity in vivo To study the protective effect of FUC on cardiotoxicity induced by DOX, the survival experiment was performed on the model of ICR mice. As shown in Fig. 1B, the single administration of DOX at the dose of 20 mg/kg produced 20% survival rate reduction in mice on day 4 after intraperitoneal injection, and the survival rate decreased to 10% on day 6. By contrast, the survival rate improved to 30%, 50% and 80% on day 6 when in combination with 125 mg/kg, 250 mg/kg, and, 500 mg/kg FUC respectively. We also examined the serum biochemical markers reflecting cardiac enzyme activity including aspartate aminotransferase

2.9. Mitochondrial membrane potential (ΔΨm) measurements The neonatal rat cardiomyocytes were cultured in 24-well plates (5 × 104/mL), after pretreatment with FUC (50 μM) for 24 h, the cells were exposed to DOX (2 μM) for next 3 h, the Δψm was measured by a 3

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(AST), lactate dehydrogenase (LDH) and creatine kinase MB (CKMB). Just as we expected, the serum cardiac enzyme activity of AST, LDH and CKMB of the DOX treated group were significantly increased when compared with the control group (p < 0.05). These increased levels of enzyme activity would be significantly attenuated by FUC treatment in a dose-dependent manner (p < 0.05) (Fig. 1C). Together, these results evidently suggest that FUC could protect against DOX-induced cardiac damage.

3.3. Fucoxanthin reduced DOX-induced the neonatal rat cardiomyocytes apoptosis The cardiotoxicity caused by DOX is multifactorial. However, myocardial cells apoptosis has been demonstrated as a common mechanism of DOX-induced myocyte loss (Semon et al., 2002), so, we subsequently examined the effect of FUC on DOX-induced cardiac myocytes apoptosis. We discovered that DOX inhibited the neonatal rat cardiomyocytes proliferation in a concentration-dependent manner. However, treatment with FUC significantly diminished the inhibition effects of DOX (Fig. 3A). To evaluate the role of apoptosis in DOX-induced cardiotoxicity, the effect of DOX on cells were carried out through DAPI staining, DNA ladder assay and Western blot analysis. As shown in Fig. 3B, DOX induced several features of apoptosis, including nuclear condensation and fragmentation in neonatal rat cardiomyocytes, while pretreatment of FUC for 24 h severe nuclear condensation and fragmentation were markedly inhibited. DNA gel electrophoresis was performed to further determine the apoptosis. A ladder pattern due to internucleosomal fragmentation of DNA was apparent when the cells were treated with 2 μM of DOX (Fig. 3F), which were markedly inhibited after pretreatment of 50 μM FUC for 24 h. Western blots showed that caspase-3, caspase-8 and PARP were upregulated and activated by DOX treatment, which could be blocked by the presence of FUC (Fig. 3C). As one early characteristic of apoptosis is the loss of mitochondrial membrane potential, the JC-1 assay was conducted with neonatal rat cardiomyocytes, and the results showed that FUC treatment could counteract the decrease of mitochondrial membrane potential induced by DOX (Fig. 3D). Additionally, we also found that FUC + DOX treatment increases the level of anti-apoptotic protein Bcl-

3.2. Fucoxanthin reversed the neonatal rat cardiomyocytes intracellular reactive oxygen species (ROS) levels caused by doxorubicin Considering that (i) several observations suggest the key role of reactive oxygen species (ROS) in DOX-induced cardiotoxicity (Doroshow, 1983; Singal, Khaper, Palace, & Kumar, 1998), (ii) fucoxanthin is implicated as a potential antioxidant for its distinct chemical structure which contains an unusual allenic bond, two hydroxyl group, and epoxide group (Sangeetha, Bhaskar, & Baskaran, 2009) (Fig. 1A), we next evaluated scavenging activity of FUC, and the changes of intracellular glutathione (GSH) and ROS levels. The results revealed that both FUC and vitamin C exhibits the scavenging activity against free radicals (Fig. 2A), as shown in Fig. 2C and B, DOX treatment caused decrease of GSH level and the increase level of ROS on the contrary. However, co-treatment with FUC could reverses DOX-induced ROS production as compared to that in DOX-only treated group, which might be involved in cardioprotective effects of FUC against oxidative damage associated with DOX.

Fig. 2. Fucoxanthin reduced the accumulation of doxorubicin induced reactive oxygen species (ROS) in primary myocardial cells. (A) DPPH radical-scavenging activity of vitamin C and fucoxanthin. Data are expressed as mean ± SD (n = 3). (B and C) Effects of FUC on DOX induced intracellular ROS levels. Myocardial cells were seeded into 24-well plates at a density of 5 × 104/ml and incubated with varying concentrations of the indicated drug, GSH level was measured by the assay kits using fluorescent spectrophotometer at 405 nm 24 h later after drug treatment (n = 3) (B), the intracellular ROS level was measured by a fluorescent dye CarboxyDCFDA using fluorescence spectrometer 3 h later after drug treatment (n = 3). *P < 0.05, **P < 0.01 compared to DOX alone group (C). 4

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Fig. 3. Fucoxanthin depressed primary myocardial cell apoptosis caused by doxorubicin. (A) MTT Assay in detecting the inhibition rate of doxorubicin combined with fucoxanthin or vitamin C on primary cardiomyocytes. Myocardial cells were seeded into 96-well plates at a density of 5 × 103 per well and treated with FUC (FUC was pretreated 24 h earlier than DOX) and DOX at the indicated concentrations. MTT assay were conducted 24 h later after DOX treatment. Values were expressed as mean ± SD (n = 3). The Data here implied that scavenging the free radicals maybe not the efficient way and the core mechanism for FUC to possess the cardioprotective effect according to the comparison with vitamin C. (B and C) The effects of FUC on DOX-induced apoptosis in primary myocardial cells. Myocardial cells were pretreated with or without FUC (50 μM) for 24 h followed by another incubation with DOX (2 μM) for 48 h, nuclei changes were observed and photographed using a fluorescence microscope (B). The expression of caspase-8, PARP and cleaved caspase-3 in whole-cell lysates were analyzed by western blot (C). (D) The effects of FUC on changes in mitochondrial membrane potential (ΔΨm) in primary myocardial cells. After preparation of appropriately treated with the indicated concentrations of FUC and DOX, the loss of ΔΨm was measured by flow cytometry using JC-1 dye. Mitochondrial depolarization is indicated by a decrease in the red/ green fluorescence intensity ratio (n = 3). *P < 0.05 compared to DOX alone group. (E) The effects of FUC on the expression Bcl-2 and Bax induced by DOX in primary myocardial cells. Myocardial cells were pretreated with or without FUC (50 μM) for 24 h followed by another incubation DOX (2 μM) for 48 h and lysed, Bcl2 and Bax were detected by western blot. (F) DNA agarose gel electrophoresis showed DNA fragmentation in cells were pretreated with or without FUC (50 μM) for 24 h followed by another incubation DOX (2 μM) for 48 h. Lanes 1, untreated; 2, treated with 2 μM DOX For 48 h; 3, pretreated with 50 μM FUC for 24 h followed by another treatment with 2 μM DOX for 48 h.; M, 100 bp DNA molecular weight marker.

2 and decreases the level of the pro-apoptotic protein Bax when compared with the DOX-only group (Fig. 3E). Thus, collectively, these results further indicated that FUC could attenuate DOX-induced cardiomyocyte mitochondria apoptosis.

2008; Matthias et al., 2014; Persons, Yazlovitskaya, & Pelling, 2000), which phosphorylate and activate p53 under stress conditions, we also detected the p-p53 level in DOX-treated cells and found an accompanying elevation in p-p53 level. Furthermore, p53 inhibitor pifithrin-α and three MAPKs specific inhibitors were employed to assess the role of p53, ERK, p38 and JNK activation in DOX-induced apoptosis in the neonatal rat cardiomyocytes. As shown in Fig. 4B, pretreatment of cells with the specific JNK inhibitor SP 600125 or p38 inhibitor SB 203580 resulted in a decrease in p53 activity as determined by the decreased p53 phosphorylation. In contrast, the levels of p-p53 were not remarkable changed when ERK inhibitor PD 98059 was applied. Thus, our results demonstrated that DOX-induced p38 and JNK activations contribute to p53 activation, which subsequently promoted apoptotic cell death. The results also showed that 10 μM SP600125 and SB203580 attenuated DOX-induced the apoptosis of cardiomyocytes and enhanced cellular viability, and also, disruption of p53 activation by 10 μM p53 inhibitor pifithrin-α led to the decrease in the DOX-induced cytotoxic and apoptotic activities. As shown in Fig. 4A, we found DOX also enhanced the expression of phosphorylated ERK. However, the specific ERK inhibitor PD98059 (10 μM) enhanced apoptosis of cells induced by DOX (Fig. 4C). These results indicate that DOX-induced the apoptosis of cardiomyocytes was triggered by the activation of p38 and JNK, while ERK pathway had a protective function against DOX-induced cell death (Fig. 4D). And these data further implicated that FUC-driven cardioprotection was dependent on the suppression of MAPK-p53 pathway activation.

3.4. Fucoxanthin affected MAPK-p53 activation and cell injury induced by doxorubicin Several previous studies have shown the implication of oxidative stress in DOX-induced cardiotoxicity, and also, some evidence indicated that employing antioxidants could counteract Dox-induced oxidative stress that results correlates with cellular injury (Šimůnek et al., 2009). In this study, the results revealed that both FUC and vitamin C exhibits the scavenging activity against free radicals (Fig. 2A), however, only FUC could protect the cardiomyocytes from the DOX-induced toxicity (Fig. 3A). Therefore, we hypothesized that there were some other mechanisms involved in DOX-driven cardiotoxicity. As illustrated in Fig. 4A, p-p38, p-ERK and p-JNK were obviously upregulated when the cardiomyocytes were exposed to DOX alone, while co-treatment with FUC could downregulated the phosphorylation of MAPK remarkably in primay cardiomyocytes. Since p53 transcriptional activity may results from the interaction with MAPK signaling pathways, in particular, p38 (Duan et al., 2011; She, Bode, Ma, Chen, & Dong, 2001; Xiao et al., 2015), JNK (Hsu, Ho, Liang, Ho, & Lee, 2010; Lorin, Pierron, Ryan, Codogno, & DjavaheriMergny, 2010; Wu, 2004), and ERK (Kaji et al., 2003; Lin, Mak, & Yang, 5

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Fig. 4. Effects of doxorubicin and fucoxanthin on the activation of p53 and MAPK in primary myocardial cells. (A) Fucoxanthin inhibits activation of the p53 and MAPK pathway induced by doxorubicin. (B) The effects of MAPK inhibitors on doxorubicininduced P53 protein activation. Cells were pre-treated for 1 h with SB203580 (SB), the p38 kinase inhibitor, or with SP600125 (SP), the JNK inhibitor, or with PD098059 (PD), the ERK inhibitorprior to being incubated for 24 h in the presence of 2 μM doxorubicin. (C) Survival fraction of doxorubicin treated neonatal rat heart myocytes, pre-treated for 1 h with SB, SP, PD or pifithrin-α (PFT-α), the p53 inhibitor, prior to incubation for 24 h in the presence of 2 μM doxorubicin and cell viability was determined by MTT Assay (n = 3). **P < 0.01 compared to control group. #P < 0.05, ##P < 0.01 compared to DOX alone group. (D) Schematic drawing representing possible protective mechanisms of fucoxanthin underlying DOX-induced apoptosis in primary myocardial cells.

3.5. Fucoxanthin does not protect cancer cell lines from doxorubicin

4. Discussions

Over the last 40 years, dexrazoxane is the only cardioprotective agent that alleviates DOX cardiotoxicity with proven efficacy in cancer patients. However, besides the potential risk of increased secondary malignancy, dexrazoxane might interfere with the antitumor efficacy of anthracyclines which could impair the clinical effect of DOX. Hence, it would be very important to evaluate the effects of cardioprotectants on the anticancer activities of anthracycline. Consequently, cytotoxicity assay was employed when DOX was combined with FUC using human leukemia cell lines K562 and U937 as models. Of interest, comparing to DOX groups; FUC augmented the cytotoxicity of DOX in a concentration-dependent manner in both cell lines. As shown in Fig. 5, the inhibition ratio of DOX was 3.9% in K562 cells at the concentrations of 0.04 μM, but increased to 35.7% and 81.3% when combined with 25 μM and 50 μM FUC. And also, the similar effect was found in U937 cells. This suggests that it could be possible to use FUC to protect the heart against DOX‐induced cardiomyocyte apoptosis but without compromising its chemopreventive effects.

Anthracyclines such as doxorubicin are among the most widely used anticancer drugs in a variety of cancer models. Yet long term cardiac damage is a serious side effect that limits the effectiveness of the drugs. The broad use of DOX for over 40 years has dramatically improved cancer survival statistics. However, life-threatening cardiac damage from DOX remains a problem. Although extensive attempts have been devoted, the exact mechanism of the DOX-induced cardiotoxicity and its progression to heart failure still remains unclear. There is an extensive literature postulating various molecular mechanisms responsible for DOX-induced cardiotoxicity such as the generation of reactive oxygen species (ROS), direct disturbance of iron homeostasis, and DNA/RNA damage (Arena et al., 1979; Shi, Moon, Dawood, Mcmanus, & Liu, 2011; Wu, Zai, et al., 2019; Wu, Zhao, et al., 2019). Among these, generation of ROS and disruption of intracellular iron homeostasis were the most widely accepted mechanistic basis for DOXinduced cardiotoxicity (Bliznakov, 1999; Joffrey et al., 2010; Wallace, 2007; Xiang et al., 2009). Although accumulating evidence suggests the involvement of oxidative stress in mediating DOX cardiotoxicity, some previous studies have showed failure of potent antioxidants and iron chelators to protect the heart against DOX provoked toxicity (Hasinoff, Fig. 5. Fucoxanthin promoted doxorubicin induced growth arrest in K562 and U937 cells. K562 (A) and U937 (B) cells were seeded into 96-well plates, after pretreatment with FUC for 24 h, cells were exposed to DOX for another 24 h, the inhibition rate was detected using MTT assay and values were expressed as mean ± SD (n = 3).

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Daywin, & Xing, 2003; Herman, Ferrans, Myers, & Van Vleet, 1985; Vejpongsa & Yeh, 2014; Van Vleet, Ferrans, & Weirich, 1980). These negative results among the effects of iron chelators on DOX-induced cardiotoxicity reveal that the intracellular iron disruption might not be the only major mechanism for the DOX cardiotoxity (Dresdale et al., 1982). Taken together, we could assume that more potential mechanisms are involved in DOX cardiotoxicity besides oxidative stresses and disturbance of iron homeostasis, which can help develop safe and novel cardioprotective agents against DOX-induced cardiotoxicity. As a manner of fact, despite of the multiple mechanisms involved in DOX-induced cardiotoxicity, cardiomyocyte apoptosis is a key event in the process of DOX induced CHF (Octavia et al., 2012; Wu, Zai, et al., 2019; Wu, Zhao, et al., 2019; Zhang, Shi, Li, & Wei, 2009). In this context, the development of novel anti-apoptotic strategies to prevent the loss of cardiac myocytes is of high importance. And the control on the apoptosis-related factors could be thought to be an efficient method for the screening of potential cardioprotectors. Since more and more evidence suggest that DOX-induced apoptosis plays an important role in its cardiotoxicity, many recent studies have focused on DOX-induced apoptotic signaling mechanism. In an effort to understand the molecular mechanisms how FUC exert cardioprotective activity, we focused attention on the MAPK signaling pathway, because (i) our previous studies suggest that FUC could counteract DOX-induced myocardial apoptosis, which might be regulated by the alternative splicing of apoptosis-associated factors, (ii) MAPKs are involved in directing cellular responses including gene expression, mitosis, proliferation, differentiation, cell survival, and apoptosis (Cao, Zhang, & Wang, 2019; Pearson et al., 2001), (iii) Many studies have shown that MAPKs were activated in cardiac tissue models in response to injury (Talmor, Applebaum, Rudich, Shapira, & Tirosh, 2000) and oxidant stress (Zanella, 2009). MAPKs pathway plays an important role in a variety of cellular responses, including proliferation, differentiation, inflammation and apoptosis, which consists of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 (Johnson & Lapadat, 2002). Previous studies reported that ROS activated JNK and p38 MAPK which are involved in apoptosis by promoting cell death rate, while the activation of ERK by growth factors and pathological stimuli is typically associated with anti-oxidative effect and cell protection (Xia, Dickens, Raingeaud, Davis, & Greenberg, 1995; Yumei et al., 2007). Therefore, we evaluated the effects of DOX treatment on the MAPKs pathway, and the results showed that phosphorylation of ERK, p38 and JNK was persistently elevated by the treatment with DOX. Consistently, evidence has also indicated that p38 and JNK were also activated in DOX-treated cardiomyocytes (Das, Ghosh, Manna, & Sil, 2011; Ghosh, Das, Manna, & Sil, 2011). Phosphorylation of ERK was rarely seen after DOX treatment in hearts in previous studies reported. However, we found DOX also leads to increased expression of phospho-ERK. Nevertheless, the specific ERK inhibitor did not reverse the apoptotic myocardial cell death induced by DOX, which indicated that ERK activation did not lead to the cardiomyocytes apoptosis (Ghosh et al., 2011). On the contrary, however, the specific inhibitors of p38 and JNK attenuated the apoptosis of cardiomyocytes, indicating the activation of p38 and JNK is responsible for DOX-induced cardiomyocytes apoptosis. In addition, we further detected the expressions of apoptosis associated genes that might contribute to DOX-induced cardiomyocytes apoptosis. And most importantly, we investigated the expressions of phosphorylated-p53 protein, which was well known for its key role as a tumor suppressor and involved in multiple central cellular processes, including genomic stability, senescence, cell cycle control, and apoptosis (Tasdemir et al., 2008). Interestingly, the present study shows that DOX markedly elevates p-p53 expression in cardiomyocytes. Consequently, we consider that activation of p38 MAPK and JNK is related to DOX-mediated apoptosis, whereas ERK may play a protective role in this process. Previous studies have reported that activation of MAPKs leads to the phosphorylation and activation of p53, resulting in further

p53-mediated downstream response (Wu, 2004). In the present study, we demonstrated that inhibition of p38 or JNK phosphorylation decreased the expression of p-p53. Thus, our results showed that DOXinduced p38 and JNK phosphorylation lead to the activation of p53, which eventually induced the cardiomyocytes apoptosis. Taken together, because the specific consequences of MAPK inhibition are similar with that executed by FUC, these clues prompted us to confirm that p38 and JNK MAPK-p53 pathways could be involved in the reduced cell toxicity effects of FUC. Furthermore, we found FUC attenuate cardiac cell death and preserve cardiac function without interfering with DOX-induced tumor cell death. The unanswered question is how MAPK-P53 inhibition can be protective in cardiac cells without protecting cancer cells from DOX. One possibility is that DOX damages cardiac and cancer cells through different mechanisms. Although much remains to be learned about the mechanisms by which FUC provide cell type-specific protection from DOX, the discovery offers compelling evidence that DOX-induced cell death can be mitigated in a cell-specific fashion. Moreover, the discovery that MAPK-P53 inhibition is sufficient for cardioprotection provides not only a potential new therapeutic target, but also a new entrance point for investigating the fundamental differences of responsiveness to DOX by cardiac and tumor cells. Future experiments will focus on elucidating the role of MAPK-P53 pathway in doxorubicin’s chemotherapeutic and cardiotoxic mechanisms. In addition, it will be informative to determine whether FUC cardioprotective effects extend to other modes of cardiac injury, such as ischemia-reperfusion or other cardiotoxic chemotherapies. 5. Conclusion In summary, we have shown for the first time that FUC, a natural product found in edible brown seaweeds, possessed the cardioprotective effect contrarious with DOX through suppressing the activation of p38, JNK and p53 in myocardial cells. Besides, FUC could enhance the anticancer activity of DOX in K562 and U937 cells, possibly because it did not activate the MAPK-p53 pathway in cancer cells. These finding thus favors FUC a potential application of DOX in oncotherapy. 6. Ethics statements All of the in vivo tests were carried by the School of Food and Pharmacy of Zhejiang Ocean University (China), which obtained the permission for performing the research protocols and all animal experiments conducted during the present study from the ethics committee of Zhejiang Ocean University. All experimental procedures were conducted under the oversight and approval of the Academy of Experimental Animal Center of Zhejiang Ocean University and in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements This research was supported by Public Service Technology Application Research Project of Science and Technology Department of Zhejiang Province (No. LGN18D060002, LGF19D060004 and LQ18D060005), National Natural Science Foundation of China (No. 81673349). References Arena, E., D'Alessandro, N., Dusonchet, L., Geraci, M., Rausa, L., & Sanguedolce, R. (1979). Repair kinetics of DNA, RNA and proteins in the tissues of mice treated with

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