Effects of Ganoderma lucidum polysaccharides against doxorubicin-induced cardiotoxicity

Biomedicine & Pharmacotherapy 95 (2017) 504–512

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Original article

Effects of Ganoderma lucidum polysaccharides against doxorubicin-induced cardiotoxicity

MARK

Fan Xua,1, Xiao Lib,1, Xu Xiaoc, Lan-fang Liua, Li Zhanga, Ping-ping Lina, Sheng-lin Zhanga, ⁎ Qing-shan Lia, a b c

Department of Oncology, Affiliated Hospital of Chengde Medical University, Chengde, 06700, Hebei, China Department of Radiology, Affiliated Hospital of Chengde Medical University, Chengde, 06700, Hebei, China Department of Pharmacy, Affiliated Hospital of Chengde Medical University, Chengde, 06700, Hebei, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Ganoderma lucidum Polysaccharides Doxorubicin Cardiotoxicity Nrf2

Doxorubicin (DOX) is a widely used anthracycline derivative anticancer drug, but the use of DOX in clinical applications is limited by its cardiotoxicity. In the current research, we were aiming to assess the effects of Ganoderma lucidum polysaccharides (GLPS) on DOX-induced cardiotoxicity and to illustrate the associated mechanisms. H9c2 rat cardiomyocytes were treated with DOX in the absence or presence of GLPS, and we found GLPS treatment ameliorated DOX-induced H9c2 cell death. Moreover, results of in vivo studies indicated that GLPS significantly decreased the serum levels of lactate dehydrogenase (LDH), creatine kinase (CK) and aspartate aminotransferase (AST) and attenuated DOX-induced histological changes of the heart tissues. In addition, we found DOX administration promoted myocardial apoptosis, potentiated oxidative stress, decreased the activities of antioxidant enzymes and increased the production of pro-inflammatory cytokines. However, GLPS pretreatment markedly attenuated all these untoward effects of DOX. Furthermore, GLPS pretreatment was found to inhibit Cul3-mediated K48-linked polyubiquitination of Nrf2 through suppressing Cul3 expression, thereby stabilizing Nrf2 expression in H9c2 cells after DOX treatment, leading to the decreased expression of P53 and p-P65 and increased levels of MDM2 and HO-1, resulted in the attenuated apoptosis, oxidative stress and inflammation induced by DOX.

1. Introduction Doxorubicin (DOX) (also referred to as Adriamycin), an anthracycline antibiotic, is well known as one of the most widely-used chemotherapeutic drugs which is used for the treatment of various cancers [1–3]. However, the clinical application of DOX has also been limited because of its potential to induce the dose-dependent cardiotoxicity [4,5]. Elucidating the mechanisms underlying cardiotoxicity induced by DOX may identify strategies to reduce cardiomyopathy risks for cancer patients. Though the specific mechanisms of DOX-induced cardiotoxicity still remain largely unknown, many studies reported that oxidative stress, cardiomyocyte apoptosis and inflammation could play important roles in DOX-induced cardiotoxicity [6–9]. Ganoderma lucidum (Reishi or Ling-Zhi) is a basidiomycete white rot fungus which is widely used in the treatment of a variety of human diseases in China for thousand years [10–12]. A variety of bioactive substances have been extracted from Ganoderma lucidum, and

⁎

1

Corresponding author. E-mail address: [email protected] (Q.-s. Li). These authors have the same contribution to this paper.

http://dx.doi.org/10.1016/j.biopha.2017.08.118 Received 16 May 2017; Received in revised form 25 August 2017; Accepted 28 August 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.

Ganoderma lucidum polysaccharides (GLPS) have been shown to be the most important material responsible for its bioactivity [13,14]. Recently, GLPS was reported to play anti-tumor, anti-oxidation, anti-virus, anti-inflammation, anti-diabetes and neuroprotection roles in various physiological processes [15–19]. However, the effect of GLPS on DOXinduced cardiotoxicity still remains largely unknown. The transcription factor NF-E2-related factor 2 (Nrf2) is one of the most important transcription factor to regulate the transcription of antioxidative genes such as heme oxygenase-1 (HO-1) [20]. Murinedouble minute 2 (MDM2) was also reported as a target gene of Nrf2, the increased expression of MDM2 induced by Nrf2 was found to contribute to the down-regulation of P53, leading to stable mitochondrial membrane potential, reduced cytochrome c release as well as inhibited caspase-3 activation therefore suppressed mitochondrial apoptosis [21]. In response to inflammatory stimuli, up-regulation of Nrf2 signaling was also found to inhibit the overproduction of cytokines and chemokines, and limited the activation of NF-ĸB signaling pathway

Biomedicine & Pharmacotherapy 95 (2017) 504–512

F. Xu et al.

were harvest for further studies.

[22]. Recently, Nrf2 was reported as an endogenous suppressor of DOXinduced cardiotoxicity by controlling both oxidative stress and autophagy in heart tissues, indicated that chemical compounds or natural pharmaceutical ingredients that are able to activate Nrf2 will be potential candidate drugs with which to treat DOX-induced cardiotoxicity [23,24]. In this research, the effect of GLPS on DOX-induced cardiotoxicity was investigated. We found GLPS treatment ameliorated cell death, mitochondrial apoptosis, oxidative stress and pro-inflammatory cytokines production in DOX-treated rats and H9c2 cells. Interestingly, we also observed that GLPS could reverse the decreased expression of Nrf2 induced by DOX, promoting Nrf2 stabilization by suppressing Cul3mediated K48-linked polyubiquitination of Nrf2, leading to the increased expression of MDM2 and HO-1, suppressing the activation of NF-κB signaling pathway. In conclusion, we suggested that GLPS as a potential candidate drug for the treatment of DOX-induced cardiotoxicity.

2.4. In vitro cell viability and anti-tumor activity Cell viability and anti-tumor activity was detected by CCK8 assay according to the manufacturer’s instructions as previously reported [25]. Briefly, cells were cultured in a 96-well plate, each well contained 1 × 104 cells in a total volume of 100 μL. After GLPS or DOX treatment, 10 μL CCK-8 was added in each well, and the optical density (OD) value was detected at 450 nm after incubating at 37 °C for 2 h. 2.5. Biochemical Assays In vivo, at the end of the experiment period, the serum samples of the rats were obtained and followed by the biochemical assays to examine the levels of LDH, CK and AST. The heart tissues were also collected to detect the content of MDA and activities of SOD, GSH, and CAT according to the kit manufacturer’s instructions. In vitro, the cell culture medium of H9c2 cells was collected for the measurement of LDH release. Moreover, the cells were harvested followed by ultrasonication. After centrifugation for 5 mins at 1000g, supernatants were collected and the levels of MDA and activities of SOD, GSH, and CAT were assessed by commercial kits.

2. Materials and methods 2.1. Reagents GLPS was obtained from Johnsun Mushroom (Hangzhou, China) and prepared as described [19]. DOX was purchased from Sigma-Aldrich (Sigma-Aldrich, MO, USA). The Cell Counting Kit-8 (CCK8) was obtained from Dojindo Laboratory (Japan). LDH, CK, AST, TNF-α, IL-6, IL-10 and MDA were measured by the kits purchased from BioLegend (San Diego, CA, USA). The commercial kits for detecting activities of the superoxide dismutase (SOD), glutathione (GSH), catalase (CAT) were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The caspase-3 activity assay kit was obtained from BioVision (Palo Alto, CA, USA) and Annexin V/PI apoptosis detection kit was produced by KEYGEN (Nanjing, China).

2.6. Histopathologic analyses At the end of the experiment period, the heart tissues were fixed in 10% buffered formalin and embedded in paraffin. Approximately 5 μm thick sections were prepared from tissue paraffin block and stained with hematoxylin and eosin (H & E). After that, pathological changes were checked by light microscopy for observation of structural abnormality (200×). The severity of changes was quantitated based on the degree of inflammation, interstitial fibrosis and myocardial disorganization. The scoring system was as follows: 0 = no damage; 1 = minimal inflammatory cell infiltration, focal minimal interstitial fibrosis and mild myocardial disorganization (less than 5%); 2 = moderate inflammatory cell infiltration, patchy multifocal interstitial fibrosis and moderate myocardial disorganization (5–20%); 3 = widespread inflammatory cell infiltration, severe interstitial fibrosis and myocardial disorganization (more than 20%) [26].

2.2. Rats and cells Male Sprague-Dawley (SD) rats weighing 220 g–250 g were purchased from the Animal Center of Peking University Health Science Center (Beijing, China). The experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals approved by the Animal Ethics Committee of the Scientific Investigation Board of Chengde Medical University. Rat cardiomyocytes H9c2, human breast cancer cells MCF-7, human hepatocellular carcinoma cells HepG2 were obtained from ATCC (Manassas, VA). All the cells were cultured at 37° C under 5% CO2 in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, and 2 mM L-glutamine.

2.7. Apoptosis assays Apoptotic cells were counted by staining with Annexin-V and propidium iodide (PI). Flow cytometry was performed to detect the percent of apoptotic cells. The caspase-3 activity of cell extracts (∼106 cells) was examined by DEVD-AMC (Peptron) according to the manufacturer’s instructions.

2.3. Experimental protocols 2.8. Quantitative PCR analysis and ELISA The rats were randomly divided into five groups: control group, GLPS (high-dose) group, DOX group, GLPS (low-dose) +DOX group, GLPS (high-dose) +DOX group (n = 15 per group). The rats in DOXtreated groups were dosed intraperitoneally with DOX (dissolved in saline, 2 mg/kg every other day for a total of 3 times). All rats received daily oral gavage with distilled water or GLPS (dissolved in distilled water, high dose: 100 mg/kg, low dose: 50 mg/kg) for 15 days prior to DOX. After 10 days of the final administration of DOX, the rats were sacrificed followed by the measurement of the body and heart weights. The heart tissues and blood samples were collected for histopathological examination, biochemical measurements and mechanism researches. For all in vitro experiments, the cells were plated at an appropriate density according to the experimental design, 24 h later, the cells were pretreated with distilled water or GLPS (dissolved in distilled water, high dose: 100 μg/ml, low dose: 50 μg/ml). Eight hours later, cells were treated with our without DOX (1 μM) for 16 h, and the cells

Heart tissues or H9c2 cells were collected at the end of the experiment period. Quantitative PCR was performed as previously reported [27]. GAPDH was used as the internal control. Primer sequences used in quantitative PCR were shown in Table 1. ELISA assays were performed to examine the production of TNF-α, IL-6, and IL-10 in serum or culture medium according to the manufacturer’s instruction. 2.9. Western blot analysis The protein extraction and western blot analysis was performed as previously described [28]. Ubiquitination assay was performed as described [27]. The antibodies for cleaved caspase-8, cleaved caspase-3, cleaved PARP, cytochrome c, MDM2, β-actin and ubiquitin (linkagespecific K48) were obtained from Abcam (Abcam, Cambridge, UK), and the other antibodies were purchased from Cell Signaling Technology 505

Biomedicine & Pharmacotherapy 95 (2017) 504–512

F. Xu et al.

3. Results

Table 1 List of primers. Number

Gene

Sequence

1

TNF-α

2

IL-6

3

IL-10

4

Nrf2

5

Cul3

6

GAPDH

Forward 5′- GACCCTCACACTCAGATCAT- 3′ Reverse 5′- TGCTACGACGTGGGCTACG- 3′ Forward 5′- TAGTCCTTCCTACCCCAACTTC- 3′ Reverse 5′- TTGGTCCTTAGCCACTCCTTC- 3′ Forward 5′- TTCCCTGGGAGAGAAGCTGA- 3′ Reverse 5′- ATGGCCTTGTAGACACCTTTGT- 3′ Forward 5′- AAGAATAAAGTCGCCGCCCA- 3′ Reverse 5′-AGATACAAGGTGCTGAGCCG- 3′ Forward 5′- ATCTGAGGTTGGTGTTGGCG- 3′ Reverse 5′-GGACTTGGGTTCTTTTGTGAGGA- 3′ Forward 5′- ATCACTGCCACTCAGAAG- 3′ Reverse 5′- AAGTCACAGGAGACAACC- 3′

3.1. GLPS prevented DOX-induced cardiomyocytes death and LDH leakage, but not inhibited the anti-tumor ability of DOX To assess the effect of GLPS on DOX-induced cardiotoxicity, we first examined the effect of GLPS on DOX-induced H9c2 rat cardiomyocytes death by CCK8 assay. As shown in Fig. 1A, H9c2 cardiomyocytes were treated with different concentrations of GLPS (25, 50, 75, 100 μg/ml) for 16 h, and we found treatment with GLPS in different concentrations had no toxicity on cell viability. In addition, we found DOX treatment decreased the cell viability, but GLPS pretreatment significantly inhibited DOX-induced decreases in living cell percent in a dose-dependent manner (Fig. 1B). The level of lactate dehydrogenase (LDH) release which is also an indicator of cell injury was also detected, and we found pretreatment with GLPS obviously prevented LDH leakage (Fig. 1C). Moreover, we examined if GLPS inhibited the anti-tumor ability of DOX, and as shown in Fig. 1D, we found DOX treatment significantly suppressed cancer cell survival. However, pretreatment with GLPS alone could also inhibit the cell ability of HepG2 cells and MCF-7 cells, which indicated that GLPS indeed has the ability of antitumor. Furthermore, we observed that GLPS did not affect the antitumor effect of DOX, and GLPS was found to improve the anti-tumor ability of DOX in a dose-dependent manner.

(Cell Signaling Technology Inc, Beverly, USA).

2.10. Statistical analysis All data are presented as mean ± S.D. of three independent experiments. Differences between the groups were analyzed by One-way ANOVA. If the ANOVA analysis was significant, Tukey multiple comparison test was used to determine the specific pairs of groups showing statistically significant differences. p < 0.05 considered statistically significant.

3.2. GLPS ameliorated DOX-induced cardiotoxicity in vivo In addition, we detected the effect of GLPS on DOX-induced cardiotoxicity in vivo. We found the rats treated with DOX showed decreased heart weight index (heart weight/body weight), but GLPS pretreated rats resulted in a dose-dependent recovery of heart weight index (Fig. 2A). The serum samples in these groups were collected, and the

Fig. 1. GLPS prevented DOX-induced cardiomyocytes death and LDH leakage, but not inhibited the anti-tumor ability of DOX. The H9c2 cardiomyocytes were treated with various concentrations of GLPS for 8 h and the cell viability was determined by CCK8 assay (A). The H9c2 cardiomyocytes were pretreated with indicated concentrations of GLPS for 8 h, followed by DOX-treatment for 16 h, cell viability (B) and LDH release (C) was examined. HepG2 and MCF-7 cells were pretreated with indicated concentration of GLPS for 8 h, followed by DOXtreatment for 16 h, the cell viability was detected. The data are representative of three independent experiments. * p < 0.05 versus DOX group, ** p < 0.01 versus DOX group, # p < 0.05 versus control group.

506

Biomedicine & Pharmacotherapy 95 (2017) 504–512

F. Xu et al.

Fig. 2. GLPS ameliorated DOX-induced cardiotoxicity in vivo. At the end of experiment period, the heart weight/body weight ratio (A), levels of LDH release (B), CK (C) and AST (D) and histological changes (E) in rat hearts were examined. Semiquantitive analysis of histopathological examination was shown (F, n = 15 per group). The data are representative of three independent experiments. * p < 0.05 versus DOX group, ** p < 0.01 versus DOX group, *** p < 0.001 versus DOX group, # p < 0.05 versus control group.

mediated internal apoptosis pathway, therefore we investigated the expression of Bax and Bcl-2 in heart tissues, and we found GLPS pretreatment inhibited Bax expression and increased the level of Bcl-2 in DOX-treated rats, resulted in an increased level of Bcl-2/Bax ratio (Fig. 3F and G).

levels of cardiac enzymes (LDH, CK and AST) in serum were evaluated to assay the cardioprotective effects of GLPS. DOX injection significantly elevated serum levels of LDH (Fig. 2B), CK (Fig. 2C) and AST (Fig. 2D); however GLPS pretreatment significantly reversed the levels of these cardiac enzymes in a dose-dependent manner. Hematoxylin and eosin (HE) staining assay was performed for the histopathological examination of heart tissues. As shown in Fig. 2E and F, GLPS pretreatment significantly ameliorated DOX-induced myocardial damage, disorganization of myofibrillar arrays, and infiltration of immune cells.

3.4. GLPS attenuated oxidative stress in vivo and in vitro Recently, increased cardiac oxidative stress was reported as the main mechanism involved in DOX-induced cardiotoxicity [28,29]. Therefore we determined the activities of antioxidant enzymes including SOD, GSH, CAT and oxidative stress indicator malondialdehyde (MDA). As shown in Fig. 4A and B, DOX treatment induced serious oxidative stress in H9c2 cells and heart tissues, as indicated by increased MDA level and suppressed the activities of SOD, GSH and CAT. However, these actions were all dose-dependently ameliorated by GLPS pretreatment.

3.3. GLPS suppressed DOX-induced cardiomyocytes apoptosis through mitochondrion-dependent apoptotic pathway We next examined the effect of GLPS on DOX-induced cardiomyocytes apoptosis. As shown in Fig. 3A and B, DOX-treatment induced obviously cell apoptosis in H9c2 cardiomyocytes, nevertheless, GLPS pretreatment significantly decreased the percent of apoptotic cells in a dose-dependent manner. In addition, the activation of caspase-3 and caspase-8 were suppressed by GLPS pretreatment (Fig. 3C and D) in H9c2 cells. Moreover, a significant release of cytochrome c from the mitochondria to the cytosol was observed in heart tissues of DOXtreated rats, as well as the increased level of cleaved poly (ADP-ribose) polymerase (PARP) (Fig. 3E). In contrast, GLPS pretreatment inhibited DOX-induced mitochondrial cytochrome c release and prevented the cleavage of PARP. Bax and Bcl-2 were essential for mitochondria-

3.5. GLPS inhibited DOX-induced pro-inflammatory cytokines production in vivo and in vitro DOX was found to induce pro-inflammatory cytokines production, leading to the excess inflammatory responses and myocardial tissue damage [30]. GLPS was also reported to play potent anti-inflammatory roles [19,31]. Therefore we evaluated the effect of GLPS on DOX507

Biomedicine & Pharmacotherapy 95 (2017) 504–512

F. Xu et al.

Fig. 3. GLPS suppressed DOX-induced cardiomyocytes apoptosis through mitochondrion-dependent apoptotic pathway. The H9c2 cardiomyocytes were pretreated with indicated concentrations of GLPS for 8 h, followed by DOX-treatment for 16 h, percent of apoptotic cells (A and B), caspase-3 activity (C) and protein levels of cleaved caspase-8 and cleaved caspase-3 (D) were detected. In vivo, at the end of experiment period, the protein levels of cleaved PARP, mitochondria and cytosolic cytochrome c (E), Bax and Bcl-2 (F) and the relative ratio of Bcl-2/Bax (G) were examined. The data are representative of three independent experiments. * p < 0.05 versus DOX group, ** p < 0.01 versus DOX group, *** p < 0.001 versus DOX group, # p < 0.05 versus control group.

degradation through 26S proteasome. With the presence of proteasome inhibitor MG132, we found that the K48-linked polyubiquitination level of Nrf2 was decreased after GLPS pretreatment (lane 2), however DOX treatment significantly increased Nrf2 K48-linked polyubiquitination (Fig. 7B). Interestingly, we also observed that pretreatment with GLPS inhibited the levels of K48-linked polyubiquitination in DOX-treated cells in a dose-dependent manner (Fig. 7B). Furthermore, we examined the protein levels of Keap1-Cul3 complex, which promoted K48-linked polyubiquitination and proteasome-dependent degradation of Nrf2 [32]. We found that treatment with GLPS had no effect on Keap1 expression and Cul3 expression was found to be increased by DOX treatment, whereas GLPS administration decreased Cul3 protein levels in the absence or presence of DOX, at the same time, we found the stabilization of Nrf2 was indeed increased after GLPS pretreatment in DOX-treated cells (Fig. 7C). Finally, we examined the mRNA levels of Cul3 and we found that GLPS inhibited the transcription of Cul3, regardless of whether DOX was involved (Fig. 7D).

induced pro-inflammatory cytokines production. We found DOX-treatment induced amounts of pro-inflammatory cytokines expression such as TNF-α and IL-6 in both mRNA and protein levels, and anti-inflammatory cytokines IL-10 expression was inhibited by DOX administration (Fig. 5A). However, pretreatment with GLPS significantly suppressed TNF-α and IL-6 production in DOX-treated H9c2 cells, and IL-10 level was also found to be reversed by GLPS pretreatment. Similar results were obtained in DOX-treated rats (Fig. 5B). 3.6. GLPS reversed the decreased expression of Nrf2 in DOX-treated cardiomyocytes Next, we examined the expression of P53 and MDM2 in DOX-treated H9c2 cardiomyocytes, we found the protein level of P53 was markedly increased, and MDM2 expression was suppressed by DOX treatment (Fig. 6A). However, GLPS pretreatment reversed the expression of P53 and MDM2 in a dose-dependent manner (Fig. 6B and C). Moreover, we found the phosphorylation of P65 was also inhibited in GLPS-pretreated H9c2 cells after DOX treatment, which indicated that the activation of NF-κB pathway was suppressed by GLPS (Fig. 6D). HO-1 was found to be the mainly regulator of oxidative stress, and we found HO-1 expression was decreased upon DOX challenge; however GLPS pretreatment reversed this action in a dose-dependent manner (Fig. 6E). Most importantly, we found the protein level of Nrf2 was decreased after DOX treatment; in contrast, GLPS pretreatment dose-dependently increased the stability of Nrf2.

4. Discussion Doxorubicin (Adriamycin) is commonly used for the treatments of solid and hematological malignancies. However, DOX does not discriminate between cancer and normal cells, therefore the use of DOX in chemotherapy has been restricted mainly due to its variety of toxicities, including cardiac, hematologic, renal, and hepatic toxicity [31]. DOXinduced cardiomyopathy always leads to the poor prognosis [33]. However, if in order to reduce cardiactoxicity and reduce the amount of DOX will seriously reduce the cure effect against cancer [34]. Therefore, novel strategies with high efficacy and safety are urgently required. Recently, traditional Chinese medicine Tongxinluo was reported to improve cardiac function in a rat model of DOX-induced dilated cardiomyopathy [35]. In addition, it was showed that QiShenYiQi pills had the ability to ameliorate DOX-induced myocardial structure damage and cardiac dysfunction in rats [36]. Moreover, total flavonoids from Clinopodium chinense (Benth.) O. Ktze (TFCC) and

3.7. GLPS suppressed Cul3 expression to stabilize Nrf2 In order to study the molecular mechanisms of GLPS on the stabilization of Nrf2, we first investigated the role of GLPS in Nrf2 mRNA expression. As shown in Fig. 7A, we found that after GLPS or DOX treatment, the transcription level of Nrf2 was not significantly changed. Therefore, we examined the K48-linked polyubiquitination levels of Nrf2, which was considered to be the main modification of protein 508

Biomedicine & Pharmacotherapy 95 (2017) 504–512

F. Xu et al.

Fig. 4. GLPS attenuated oxidative stress in vivo and in vitro. At the end of experiment period, the MDA content, activities of SOD, GSH and CAT in H9c2 cells (A) or heart tissues of rats (B) were examined. The data are representative of three independent experiments. * p < 0.05 versus DOX group, ** p < 0.01 versus DOX group, *** p < 0.001 versus DOX group, # p < 0.05 versus control group.

years and it has an extensive variety of pharmacological activities responsible for its health benefits such as anti-oxidant, anti-cancer and anti-inflammatory activities [10]. Previous evidence showed that polysaccharides extracted from G. lucidum are the main source of the biological activities and therapeutic uses of G. lucidum [13]. In the present study, as an extension of function of GLPS, we were aiming to investigate the effect of GLPS on DOX-induced cardiotoxicity. Apoptosis, oxidative stress and inflammation are important manifestations of DOX-induced cytotoxicity, therefore we examined the effect of GLPS on these physiological processes. We found pretreatment with GLPS inhibited DOX-induced cell apoptosis, and the inhibition effect may related to the mitochondria-dependent internal apoptosis pathway as indicated by the modification of cytochrome c and Bcl-2/Bax ratio. We

myricitrin were found to protect against DOX-induced cardiotoxicity without influence of anti-tumor ability of DOX [6,28]. In the current study, we investigated the effect of GLPS on DOX-induced cardiotoxicity, and we found GLPS greatly ameliorated DOX-induced H9c2 cardiomyocytes death and myocardial structure damage, as well as the decreased levels of LDH, CK and AST. Interestingly, we found that GLPS did not affect the anti-tumor effect of DOX, and this effect was observed to be improved by GLPS pretreatment. Taken together with the reported studies about anti-cancer ability of GLPS [37,38], we believed that the combinative strategy of DOX and GLPS in the treatment of cancer could have a promising future. Ganoderma lucidum, is more than just an ordinary fungus; it has been widely used as a traditional Chinese medicine for more than 2000 509

Biomedicine & Pharmacotherapy 95 (2017) 504–512

F. Xu et al.

Fig. 5. GLPS inhibited DOX-induced proinflammatory cytokines production in vivo and in vitro. At the end of experiment period, mRNA and protein levels of TNF-α, IL-6 and IL-10 in H9c2 cells (A) or heart tissues of rats (B) were detected. The data are representative of three independent experiments. * p < 0.05 versus DOX group, ** p < 0.01 versus DOX group, *** p < 0.001 versus DOX group, # p < 0.05 versus control group.

Fig. 6. GLPS reversed the decreased expression of Nrf2 in DOX-treated cardiomyocytes. The H9c2 cardiomyocytes were pretreated with indicated concentrations of GLPS for 8 h, followed by DOX-treatment for 16 h, the protein levels of P53, MDM2, p-P65, P65, HO-1, Nrf2 and β-actin were detected (A). Relative expression of P53 (B), MDM2 (C), p-P65 (D), HO-1(E) and Nrf2 (F) in (A) was assessed. The data are representative of three independent experiments. * p < 0.05 versus DOX group, ** p < 0.01 versus DOX group, *** p < 0.001 versus DOX group, # p < 0.05 versus control group.

510

Biomedicine & Pharmacotherapy 95 (2017) 504–512

F. Xu et al.

Fig. 7. GLPS suppressed Cul3 expression to stabilize Nrf2. The H9c2 cardiomyocytes were pretreated with indicated concentrations of GLPS for 8 h, followed by DOXtreatment for 16 h, the mRNA levels of Nrf2 was examined (A). K48-linked polyubiquitination levels of Nrf2 (B) were detected by western blot with the presence of proteasome inhibitor MG132. Protein levels of Keap1, Cul3 and Nrf2 after GLPS pretreatment in DOX-treated H9c2 cells were examined (C). Relative mRNA levels of Cul3 were measured (D). The data are representative of three independent experiments. ** p < 0.01 versus DOX group, *** p < 0.001 versus DOX group, # p < 0.05 versus control group.

dose-dependent manner. In resting state, Keap1 binds to Nrf2 in the cytoplasm and recruits E3 ligase Cul3 for K48-linked polyubiquitination of Nrf2 to cause Nrf2 degradation. When oxidative stress and other stimuli lead to conformational changes in Keap1, Nrf2 is released and transferred to the nucleus to initiate transcription of target genes [32]. In the current study, we found GLPS pretreatment inhibited K48-linked polyubiquitination of Nrf2 in the absence or presence of DOX, in addition, we observed that pretreatment with GLPS significantly decreased the protein levels of Cul3, which may associate with the attenuated levels of Nrf2 K48-linked polyubiquitination. Finally, we also found GLPS pretreatment significantly inhibited Cul3 mRNA transcription, however, the precise mechanisms that how GLPS suppressed Cul3 mRNA expression still need further studies. It is noteworthy that we also found that the expression of Cul3 was reduced and the ubiquitination of Nrf2 was also attenuated after the addition of GLPS alone, but did not affect a series of physiological processes. We speculated that this might be due to the fact that GLPS only affect Cul3 mRNA transcription with or without DOX added, but did not alter the modification and conformation of Keap1, which was not sufficient to allow Nrf2 to dissociate and transfer into the nucleus. However, when DOX was added, the conformation of Keap1 was changed, Nrf2 could be transferred into the nucleus and the downstream gene transcription was initiated. In conclusion, our present study provided the evidence that GLPS could inhibited DOX-induced cardiomyocytes death, apoptosis, oxidative stress and pro-inflammatory cytokines production, therefore ameliorated cardiotoxicity induced by DOX, and we found GLPS pretreatment stabilized Nrf2 expression through suppressing Cul3-mediated K48-linked polyubiquitination of Nrf2, leading to the modification of MDM2, P53, HO-1 and inhibition of NF-κB signaling pathway.

observed that GLPS pretreatment significantly inhibited DOX-induced oxidative stress and pro-inflammatory cytokines production, and GLPS was also found to increase the level of anti-oxidative enzymes and antiinflammatory cytokines expression both in vivo and in vitro. Mitochondria-mediated internal apoptosis was found to occur in P53-dependent manner in cardiomyocytes [39], and MDM2 was shown to promote proteasomal degradation of P53 [21]. We wondered if GLPS inhibits apoptosis through this pathway, so we detected the protein levels of P53 and MDM2, and we found MDM2 expression was induced by GLPS in DOX-treated H9c2 cardiomyocytes, consistently, protein level of P53 was decreased. We also suspected how GLPS prevent oxidative stress after DOX treatment. Cardiac HO-1 was reported as a crucial regulator of DOX-induced oxidative stress in cardiomyocytes [40], and in the present study, we found the expression of HO-1 was significantly decreased after DOX treatment, but the expression was reversed by GLPS pretreatment. Results from several studies have indicated that increased intracellular oxygen radicals mediated by DOX induce inflammation [41], indeed, we found DOX treatment induced amounts of pro-inflammatory cytokines production and activation of NF-κB pathway, and the anti-inflammatory cytokine such as IL-10 was decreased. However, GLPS pretreatment significantly reduced the production of pro-inflammatory cytokines and suppressed the phosphorylation of P65, consistent with the reported anti-inflammatory effect of GLPS in previous researches, and the IL-10 was increased which may related to the reversed expression of HO-1. Furthermore, we wonder if GLPS could regulate apoptosis, oxidative stress and inflammation through Nrf2, which was found to be a critical regulator of these processes, and previous study also showed that Nrf2 deficiency could exaggerate DOX-induced cardiotoxicity and cardiac dysfunction [25]. Indeed, we found Nrf2 was significantly reduced after DOX treatment, but pretreatment with GLPS greatly improved the stability of Nrf2 in a 511

Biomedicine & Pharmacotherapy 95 (2017) 504–512

F. Xu et al.

Immunopharmacol. 25 (2015) 112–120. [20] T.W. Kensler, N. Wakabayashi, S. Biswal, Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 89–116. [21] J.H. Hagemann, D. Thomasova, S.R. Mulay, H.J. Anders, Nrf2 signalling promotes ex vivo tubular epithelial cell survival and regeneration via murine double minute (MDM)-2, Nephrol. Dial. Transplant. 28 (2013) 2028–2037. [22] H.O. Pae, G.S. Oh, B.S. Lee, J.S. Rim, Y.M. Kim, H.T. Chung, 3-Hydroxyanthranilic acid, one of L-tryptophan metabolites, inhibits monocyte chemoattractant protein-1 secretion and vascular cell adhesion molecule-1 expression via heme oxygenase-1 induction in human umbilical vein endothelial cells, Atherosclerosis 187 (2006) 274–284. [23] S. Li, W. Wang, T. Niu, H. Wang, B. Li, L. Shao, et al., Nrf2 deficiency exaggerates doxorubicin-induced cardiotoxicity and cardiac dysfunction, Oxid. Med. Cell. Longevity 2014 (2014) 748524. [24] L.F. Wang, S.W. Su, L. Wang, G.Q. Zhang, R. Zhang, Y.J. Niu, et al., Tert-butylhydroquinone ameliorates doxorubicin-induced cardiotoxicity by activating Nrf2 and inducing the expression of its target genes, Am. J. Transl. Res. 7 (2015) 1724–1735. [25] Y. Wei, D. Liu, X. Jin, P. Gao, Q. Wang, J. Zhang, et al., PA-MSHA inhibits the growth of doxorubicin-resistant MCF-7/ADR human breast cancer cells by downregulating Nrf2/p62, Cancer Med. 5 (2016) 3520–3531. [26] Y. Selcoki, E. Uz, R. Bayrak, S. Sahin, A. Kaya, B. Uz, et al., The protective effect of erdosteine against cyclosporine A-induced cardiotoxicity in rats, Toxicology 239 (2007) 53–59. [27] Q.Z. Kong, L.T. Guo, J.N. Yang, Y.F. Wang, J.X. Zhao, S.H. Kong, et al., Anti-inflammatory effects of TRAF-Interacting protein in rheumatoid arthritis fibroblastlike synoviocytes, Mediators Inflamm. 2016 (2016) 3906108. [28] J. Sun, G. Sun, X. Cui, X. Meng, M. Qin, Sun X. Myricitrin, Protects against doxorubicin-induced cardiotoxicity by counteracting oxidative stress and inhibiting mitochondrial apoptosis via ERK/P53 pathway, Evid. Based Complement. Altern. Med. 2016 (2016) 6093783. [29] H. Wang, P. Yu, H. Gou, J. Zhang, M. Zhu, Z.H. Wang, et al., Cardioprotective effects of 20(S)-Ginsenoside rh2 against doxorubicin-Induced cardiotoxicity in vitro and In vivo, Evid. Based Complement. Altern. Med. 2012 (2012) 506214. [30] N.M. Elsherbiny, M.F. Salama, E. Said, M. El-Sherbiny, M.M. Al-Gayyar, Crocin protects against Doxorubicin-induced myocardial toxicity in rats through downregulation of inflammatory and apoptic pathways, Chem. Biol. Interact. 247 (2016) 85. [31] R. Injac, M. Boskovic, M. Perse, E. Koprivecfurlan, A. Cerar, A. Djordjevic, et al., Acute doxorubicin nephrotoxicity in rats with malignant neoplasm can be successfully treated with fullerenol C60(OH)24 via suppression of oxidative stress, Pharmacol. Rep. Pr 60 (2008) 742. [32] M. Mcmahon, K. Itoh, M. Yamamoto, J.D. Hayes, Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression, J. Biol. Chem. 278 (2003) 21592–21600. [33] K. Chatterjee, J. Zhang, R. Tao, N. Honbo, J.S. Karliner, Vincristine attenuates doxorubicin cardiotoxicity, Biochem. Biophys. Res. Commun. 373 (2008) 555–560. [34] L.D. Hole, T.H. Larsen, K.O. Fossan, F. Lime, J. Schjott, Diazoxide protects against doxorubicin-induced cardiotoxicity in the rat, BMC Pharmacol. Toxicol. 15 (2014) 28. [35] F.F. Shen, T.H. Jiang, J.Q. Jiang, Y. Lou, X.M. Hou, Traditional chinese medicine tongxinluo improves cardiac function of rats with dilated cardiomyopathy, Evid. Based Complement. Altern. Med. 2014 (2014) 323870. [36] D.X. Tang, H.P. Zhao, C.S. Pan, Y.Y. Liu, X.H. Wei, X.Y. Yang, et al., QiShenYiQi pills, a compound chinese medicine, ameliorates doxorubicin-Induced myocardial structure damage and cardiac dysfunction in rats, Evid. Based Complement. Altern. Med. 2013 (2013) 480597. [37] A. Li, X. Shuai, Z. Jia, H. Li, X. Liang, D. Su, et al., Ganoderma lucidum polysaccharide extract inhibits hepatocellular carcinoma growth by downregulating regulatory T cells accumulation and function by inducing microRNA-125b, J. Transl. Med. 13 (2015) 100. [38] Y. Chai, G. Wang, L. Fan, M. Zhao, A proteomic analysis of mushroom polysaccharide-treated HepG2 cells, Sci. Rep. 6 (2016) 23565. [39] R. Kang, D. Tang, M.T. Loze, H.J. Zeh, Apoptosis to autophagy switch triggered by the MHC class III-encoded receptor for advanced glycation endproducts (RAGE), Autophagy 7 (2011) 91–93. [40] J.S. Koh, C.O. Yi, R.W. Heo, J.W. Ahn, J.R. Park, J.E. Lee, et al., Protective effect of cilostazol against doxorubicin-induced cardiomyopathy in mice, Free Radic. Biol. Med. 89 (2015) 54–61. [41] P. Mukhopadhyay, M. Rajesh, S. Batkai, Y. Kashiwaya, G. Hasko, L. Liaudet, et al., Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro, Am. J. Physiol. Heart Circ. Physiol. 296 (2009) H1466–83.

Conflict of interests The authors declare that there is no conflict of interests. References [1] Y.I. Cho, S. Park, S.Y. Jeong, H.S. Yoo, In vivo and in vitro anti-cancer activity of thermo-sensitive and photo-crosslinkable doxorubicin hydrogels composed of chitosan-doxorubicin conjugates, Eur. J. Pharm. Biopharm. 73 (2009) 59–65. [2] K. Effenberger-Neidnicht, R. Schobert, Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin, Cancer Chemother. Pharmacol. 67 (2011) 867–874. [3] J. Gu, W. Hu, Z.P. Song, Y.G. Chen, D.D. Zhang, C.Q. Wang, Resveratrol-induced autophagy promotes survival and attenuates doxorubicin-induced cardiotoxicity, Int. Immunopharmacol. 32 (2016) 1–7. [4] L.A. Smith, V.R. Cornelius, C.J. Plummer, G. Levitt, M. Verrill, P. Canney, et al., Cardiotoxicity of anthracycline agents for the treatment of cancer: systematic review and meta-analysis of randomised controlled trials, BMC Cancer 10 (2010) 337. [5] G. Takemura, H. Fujiwara, Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management, Prog. Cardiovasc. Dis. 49 (2007) 330–352. [6] R.C. Chen, X.D. Xu, X. Zhi Liu, G.B. Sun, Y.D. Zhu, X. Dong, et al., Total flavonoids from clinopodium chinense (Benth.) O. ktze protect against doxorubicin-induced cardiotoxicity in vitro and In vivo, Evid. Based Complement. Altern. Med. 2015 (2015) 472565. [7] R. Guo, K. Wu, J. Chen, L. Mo, X. Hua, D. Zheng, et al., Exogenous hydrogen sulfide protects against doxorubicin-induced inflammation and cytotoxicity by inhibiting p38MAPK/NFkappaB pathway in H9c2 cardiac cells, Cell Physiol. Biochem. 32 (2013) 1668–1680. [8] B.D. Sahu, J.M. Kumar, M. Kuncha, R.M. Borkar, R. Srinivas, R. Sistla, Baicalein alleviates doxorubicin-induced cardiotoxicity via suppression of myocardial oxidative stress and apoptosis in mice, Life Sci. 144 (2016) 8–18. [9] N. Razmaraii, H. Babaei, A. Mohajjel Nayebi, G. Assadnassab, J. Ashrafi Helan, Y. Azarmi, Crocin treatment prevents doxorubicin-induced cardiotoxicity in rats, Life Sci. 157 (2016) 145–151. [10] B.S. Sanodiya, G.S. Thakur, R.K. Baghel, G.B. Prasad, P.S. Bisen, Ganoderma lucidum: a potent pharmacological macrofungus, Curr. Pharm. Biotechnol. 10 (2009) 717–742. [11] M. Xuan, M. Okazaki, N. Iwata, S. Asano, S. Kamiuchi, H. Matsuzaki, et al., Chronic treatment with a water-soluble extract from the culture medium of ganoderma lucidum mycelia prevents apoptosis and necroptosis in hypoxia/ischemia-induced injury of type 2 diabetic mouse brain, Evid. Based Complement. Altern. Med. 2015 (2015) 865986. [12] H.J. Lin, Y.S. Chang, L.H. Lin, C.F. Haung, C.Y. Wu, K.L. Ou, An immunomodulatory protein (Ling zhi-8) from a ganoderma lucidum induced acceleration of wound healing in rat liver tissues after monopolar electrosurgery, Evid. Based Complement. Altern. Med. 2014 (2014) 916531. [13] J.W. Hsu, H.C. Huang, S.T. Chen, C.H. Wong, H.F. Juan, Ganoderma lucidum polysaccharides induce macrophage-Like differentiation in human leukemia THP1Cells via caspase and p53 activation, Evid. Based Complement. Altern. Med. 2011 (2011) 358717. [14] R.R. Paterson, Ganoderma − a therapeutic fungal biofactory, Phytochemistry 67 (2006) 1985–2001. [15] S.A. Heleno, L. Barros, A. Martins, M.J.R.P. Queiroz, C. Santos-Buelga, I.C.F.R. Ferreira, Fruiting body, spores and in vitro produced mycelium of Ganoderma lucidum from Northeast Portugal: a comparative study of the antioxidant potential of phenolic and polysaccharidic extracts, Food Res. Int. 46 (2012) 135–140. [16] J. Habijanic, M. Berovic, B. Boh, M. Plankl, B. Wraber, Submerged cultivation of Ganoderma lucidum and the effects of its polysaccharides on the production of human cytokines TNF-alpha, IL-12, IFN-gamma, IL-2, IL-4, IL-10 and IL-17, New Biotechnol. 32 (2015) 85–95. [17] C.H. Wang, S.C. Hsieh, H.J. Wang, M.L. Chen, B.F. Lin, B.H. Chiang, et al., Concentration variation and molecular characteristics of soluble (1,3;1,6)-beta-Dglucans in submerged cultivation products of Ganoderma lucidum mycelium, J. Agric. Food Chem. 62 (2014) 634–641. [18] R. Hanaoka, Y. Ueno, S. Tanaka, K. Nagai, T. Onitake, K. Yoshioka, et al., The waterSoluble extract from cultured medium of ganoderma lucidum (Reishi) mycelia (Designated as MAK) ameliorates murine colitis induced by trinitrobenzene sulphonic acid, Scand. J. Immunol. 74 (2011) 454–462. [19] Y.J. Liu, J.L. Du, L.P. Cao, R. Jia, Y.J. Shen, C.Y. Zhao, et al., Anti-inflammatory and hepatoprotective effects of Ganoderma lucidum polysaccharides on carbon tetrachloride-induced hepatocyte damage in common carp (Cyprinus carpio L.), Int.

512