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Cryptotanshinone protects against adriamycin-induced mitochondrial dysfunction in cardiomyocytes a
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Yanshan Zhang , Liang Chen , Fan Li , Huijuan Wang , Yunyi Yao , Jiamei Shu & Ming-Zhong c
Ying a
Department of Tumor Surgery, Wuwei Tumor Hospital, Wuwei, Gansu PR China,
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Department of Paediatrics, Changhai Hospital, Second Military Medical University, Shanghai, PR China, c
International Medical Center, Chinese PLA General Hospital, Beijing PR China,
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Department of Tumor Chemotherapy, Wuwei Tumor Hospital, Wuwei, Gansu, PR China,
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Research Center for Biochemistry and Molecular Biology, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu, PR China, and f
Department of Cardiology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu PR China Published online: 05 May 2015.
To cite this article: Yanshan Zhang, Liang Chen, Fan Li, Huijuan Wang, Yunyi Yao, Jiamei Shu & Ming-Zhong Ying (2015): Cryptotanshinone protects against adriamycin-induced mitochondrial dysfunction in cardiomyocytes, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1029052
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http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–6 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1029052
ORIGINAL ARTICLE
Cryptotanshinone protects against adriamycin-induced mitochondrial dysfunction in cardiomyocytes Yanshan Zhang1*, Liang Chen2*, Fan Li3, Huijuan Wang4, Yunyi Yao5, Jiamei Shu6, and Ming-Zhong Ying3 Department of Tumor Surgery, Wuwei Tumor Hospital, Wuwei, Gansu, PR China, 2Department of Paediatrics, Changhai Hospital, Second Military Medical University, Shanghai, PR China, 3International Medical Center, Chinese PLA General Hospital, Beijing, PR China, 4Department of Tumor Chemotherapy, Wuwei Tumor Hospital, Wuwei, Gansu, PR China, 5Research Center for Biochemistry and Molecular Biology, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu, PR China, and 6Department of Cardiology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, PR China
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Abstract
Keywords
Context: The serious side effect of Adriamycin (ADR) is cardiomyopathy. Cryptotanshinone (CRY) is widely and safely used as antioxidant with MTD more than 5 mg/g in rats (p.o). Objective: The objective of this study is to study the protection effects of CRY against ADR-induced mitochondrial dysfunction in cardiomyocytes. Materials and methods: The chemical administration lasted for 20 days with an effective dose of CRY (p.o.) at 50 mg/kg in rats. Mitochondrial respiratory chain complex activities, ATP generation, mitochondrial membrane potential (MMP), superoxide anion free radical, oxidative stress-relative enzymes, and mitochondrial biogenesis-relative factors in normal control, ADR (i.p., 1.25 mg/kg), and ADR (i.p., 1.25 mg/kg) + CYP (p.o., 50 mg/kg) groups were detected. Results: 50 mg/kg CRY significantly promoted the energy production of ATP (16.99 ± 2.38 nmol/g Pro) (Pro: Protein) by increasing the complexes activities except II (p40.05). After the treatment of CRY, the suppressed MMP was increased while superoxide anion free radical (0.57 ± 0.07/mg Pro) was inhibited markedly. Mitochondrial biogenesis-relative factors PGC-1a, NRF-1, and TFAM were also promoted. Remarkable augmentations of NO, inducible nitric oxide synthase (iNOS), and increased activity of GSH-PX (p50.05) were also detected after the treatment of CRY, while no obvious changes on the activity of nitric oxide synthase (cNOS; p40.05) were observed. Discussion and conclusion: These results suggest that CRY protects against ADR-induced mitochondrial dysfunction in cardiomyocytes. It could be an ideal potential drug of cardioprotection.
ATP generation, cardiovascular protection, mitochondrial biogenesis, mitochondrial complex activity, mitochondrial membrane potential, mitochondrial respiratory chain, oxidative stress, superoxide anion free radical
Introduction Adriamycin (ADR), also known as doxorubicin, is an anthracycline antibiotic drug for cancer chemotherapy by intercalating DNA of cancer cells. It is widely used as the first-line drug in the treatments of hematological malignancies, types of carcinoma, and soft tissue sarcomas. The most serious adverse effect is cardiomyopathy, leading to congestive heart failure (Octavia et al., 2012). Few effective drugs can be used to prevent the cardiotoxicity of ADR. Cryptotanshinone (CRY) is one of the most abundant constituents of the root of Salvia miltiorrhiza Bunge (Lamiaceae). It is widely used as a medical herb in Asian and European countries for the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the major bioactive compounds of Salvia miltiorrhiza roots including the *These authors contributed equally to this work. Correspondence: Prof. Ming-Zhong Ying, International Medical Center, Chinese PLA General Hospital, Beijing 100853, PR China. E-mail: [email protected]
History Received 12 August 2014 Revised 3 March 2015 Accepted 09 March 2015 Published online 10 April 2015
cryptotanshinone, are diterpene quinone, promoting the cardiovascular protection (Han et al., 2008). Unlike tanshinone I and tanshinone IIA, fewer studies of cryptotanshinone have been focused on the pharmacological effects including the cardiovascular protection. Research indicates ADRinduced cardiotoxicity via mitochondria (Carvalho et al., 2014; Finsterer & Ohnsorge, 2013). Given that CRY can adjust the oxidative stress (Chen et al., 2014), which is caused by mitochondria, this research mainly focused on the protection on cardiomyocytes via mitochondria instead of other organelles. Our previous research indicates that CRY is a safe chemical with no cytotoxicity or oncogenicity. The ED50 of CRY is 10 mg/kg in rats for cardioprotection according to our previous research. The effect is dosedependent. The maximum tolerated dose (MTD) of CRY is even more than 5 mg/g (p.o) in rats. Our former study tested the dose at 10, 20, 50, 100 mg/kg and indicated 50 and 100 mg/kg were the most effective doses for cardioprotection. No obvious differences were found between 50 and 100 mg/kg. The purpose of this study was to examine whether
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mitochondrial dysfunction could be relieved by CRY and the cardiovascular protection of CRY against ADR-induced mitochondrial dysfunction in rat cardiomyocytes.
Materials and methods
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Animal model generation and drug treatment Adult male Wistar rats weighing 300 ± 50 g (Beijing HFK Bioscience Co., LTD, Beijing, China) were assigned into three groups randomly, six rats each group: the normal control (NC) group, the ADR group, and the ADR + CRY group. Rats in the NC group were treated with 0.9% normal saline (i.p.) and 0.5% sodium carboxymethylcellulose (CMC-Na, p.o.) for a total of 20 d. Rats in the ADR group were treated with 0.9% normal saline (i.p.) and 0.5% CMC-Na (p.o.) for a total of 20 d. On the 9th day, rats were treated with 1.25 mg/kg ADR (i.p.) every 2 d, six times for a total of 12 d (2 d 6 doses). Our previous research found that 50 mg/kg CRY (p.o.) was an effective dose for cardioprotection in rats. Rats in the ADR+CRY group were treated with normal saline (i.p.) and CRY (dissolved in 0.5% CMC-Na, p.o., 50 mg/kg) for a total of 20 d. On the 9th day, rats were treated with 1.25 mg/kg of ADR (i.p.) every 2 d, six times for a total of 12 d (2 d 6 doses). Rats were sacrificed and myocardium was isolated on the 21st day. Mitochondria were purified by Percoll density gradient centrifugation according to a published paper (Morin et al., 2002). In accordance with the published guidelines of the China Council on Animal Care, this research was approved by the Medical Ethics Committee of Chinese PLA General Hospital and Wuwei Tumor Hospital. Determination of mitochondrial respiratory chain complexes activities and ATP generation The activities of mitochondrial respiratory chain complexes, complex I (NADH dehydrogenase, kit Cat.: A089-1), complex II (succinate dehydrogenase, kit Cat.: A089-2), complex III (ubiquinol cytochrome c reductase, kit Cat.: A089-3), and complex IV (cytochrome c oxidase, kit Cat.: A089-4) were analyzed using ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the instructions of the manufacturer. Briefly, blank wells, standard wells, and testing sample wells were set separately. Sample dilution (40 ll) was added into testing sample wells, then 10 ll testing sample was added (final dilution is 1/5), mixed gently, incubated at 37 C for 30 min, and then washed for five times. HRP-conjugate reagent (50 ml) was added to each well except blank well, incubated at 37 C for 30 min, and then washed. Chromogen solution A (50 ll) and chromogen solution B (50 ll) were added into each well for 15 min at 37 C and then stop solution (50 ll) was added into each well to stop the reaction for 15 min, waiting for the blue to change yellow. Blank wells were adjusted as 0. The absorbance in each well was recorded (k ¼ 450 nm). The value in the NC group was set as 1 while other relative values were calculated by comparison. The adenosine triphosphate (ATP) concentration in the mitochondria was measured using reverse-phase highpressure liquid chromatography (Agilent Technologies, Palo Alto, CA) following the protocol reported previously (Chen et al., 2005).
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Determination of mitochondrial membrane potential and superoxide anion free radical Mitochondrial membrane potential (MMP) was detected using 5,50 ,6,60 -Tetrachloro-1,10 ,3,30 -tetraethyl-imidacarbocyanine iodide (JC-1) staining kit (C2006, Beyotime Institute of Biotechnology, Shanghai, China). About 30 mg mitochondria sample (100 mL) was incubated with 900 mL JC-1 staining buffer. The fluorescence intensity was detected in the fluorescence spectrophotometer (F-7000, Hitachi, Tokyo, Japan) at 37 C for 20 min. The ratio of aggregates (k ¼ 590 nm) to monomer (k ¼ 530 nm) was calculated as an indicator of MMP. The value in the NC group was set as 1 while other relative values were calculated by comparison. The superoxide anion free radical was detected using a kit according to the instruction of the manufacturer (Nanjing Jiancheng Bioengineering Institute, China. kit Cat.: A052). Determination of mitochondrial biogenesis-relative factors The total RNA was isolated from myocardium in NC, ADR, and ADR + CRY groups and was reverse transcripted into cDNA as the templates for the Real-time qPCR detection (ABI7500, Applied Biosystems, Waltham, MA). Oligonucleotide primers for mitochondrial biogenesis-relative genes were peroxisome proliferator-activated receptor-g-coactivator 1-a (PGC-1a): up: 50 -TATTCCAGGTCAAGATCAA GGTCC-30 , down: 50 -CTTTCGTGCTCATTGGCTTCATAG30 , 185 bp; nuclear respiratory factor 1 (NRF-1): up: 50 -CAGA TAGTCCTGTCTGGGGAAACC-30 , down: 50 -CCGCCATAA TGAATCCCTTTCCAA-30 , 169 bp, mitochondrial transcription factor A (TFAM): up: 50 -CCATGGACTTCTGCCCAC TGAAT-30 , down: 50 -CTTCACAAACCCGCACGAAACTG30 , 204 bp, (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China). Total protein extracted (30 mg) from in NC, ADR, and ADR + CRY groups was boiled with 4 loading buffer at 100 C for 5 min and then injected to 12.5% SDS-PAGE. After electrophoresis, it was transferred to the PVDF membrane (Invitrogen, Waltham, MA), blocked in 5% fat-free milk for 1 h and incubated overnight at 4 C with anti-PGC-1 (K-15): (goat polyclonal IgG, sc-5816, 91 kDa, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, 1:600), anti-NRF-1 (rabbit monoclonal IgG, ab175932, 54 kDa, Abcam, Cambridge, MA, 1:1000), and anti-TFAM (rabbit polyclonal IgG, Catalog# 3885-100, 27 kDa, BioVision, Inc., Milpitas, CA, 1:800) and then secondary antibody for 1 h at room temperature. b-Actin was used as the native control. The value (target/b-actin) in the NC group was set as 1 while the relative data were calculated by comparing with the NC group. Determination of oxidative stress-relative enzymes Myocardium of rats in three groups were isolated and used for the detection of activities of oxidative stress-relative enzymes nitric oxide (NO) (A013-2), constitutive nitric oxide synthase (cNOS), and inducible nitric oxide synthase (iNOS) (A014-1), glutathione peroxidase (GSH-PX) (A005). Kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Chemical colorimetry was used for the detection in spectrophotometer.
DOI: 10.3109/13880209.2015.1029052
Statistical analysis All data were expressed as mean ± SD and analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL). The results were evaluated by one-way ANOVA. Statistical significances were defined as p50.05(*) and p50.01(**).
Results
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Effects of ADR on mitochondrial respiratory chain complexes activities and ATP generation Due to the toxicity of ADR on myocardium of rat, the activities of mitochondrial complexes I (46.68 ± 4.73), II (60.03 ± 7.51), III (44.30 ± 7.46), and IV (49.27 ± 5.02) in the ADR group were all greatly inhibited. Except complex II (67.31 ± 7.10, p40.05), remarkable augmentations of the activities of complexes I (65.14 ± 9.21), III (58.73 ± 4.48), and IV (63.29 ± 7.04) were detected after the treatment of CRY, despite lower than normal. Data are shown in Figure 1(A). The activities of complexes regulate the ATP generation. Low activities of mitochondrial complexes will result in the suppressed ATP generation. The ATP generation was normal in the NC group (36.48 ± 5.79 nmol/g) Prowhile greatly inhibited in the ADR group (8.90 ± 2.36 nmol/g Pro). After the treatment of CRY, the ATP generation (16.99 ± 2.38 nmol/g Pro) was markedly promoted, despite lower than normal. These results suggested the functional recovery of ETC by CRY. Taken together, these results indicated that CRY promoted the ATP generation through the activity recoveries of complexes I, III, and IV. Data are shown in Figure 1(B). Effects of ADR on the mitochondrial membrane potential and superoxide anion free radical CRY could promote the structural repair and functional recovery on mitochondrial membrane. Due to the damage of ADR on mitochondria, the MMP was markedly decreased in the ADR group (0.61 ± 0.08). After the treatment of CRY, it was significantly increased (0.78 ± 0.07), despite lower than normal. Data are shown in Figure 2(A). As an indicator of oxidative stress, the release of superoxide anion free radical
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was markedly increased in the ADR group (0.75 ± 0.08/mg Pro) compared with the NC group (0.26 ± 0.06/mg Pro). After the treatment of CRY, it was significantly inhibited (0.57 ± 0.07/mg Pro). The damage on mitochondria caused by oxidative stress could also be weakened after the inhibition. Data are shown in Figure 2(B).
Effects of ADR on mitochondrial biogenesis-relative factors The function of mitochondrial biogenesis was weakened because of the toxicity of ADR on mitochondria. On the gene level, ADR down-regulated the expressions of mitochondrial biogenesis-relative factors PGC-1a (0.57 ± 0.06), NRF-1 (0.54 ± 0.09), and TFAM (0.60 ± 0.10). The expressions of PGC-1a (0.72 ± 0.07), NRF-1 (0.75 ± 0.08), and TFAM (0.80 ± 0.07) were increased significantly after the treatment of CRY, despite lower than the NC group. On the protein level, PGC-1a (0.58 ± 0.07), NRF-1 (0.41 ± 0.07), and TFAM (0.53 ± 0.08) were also down-regulated in the ADR group. CRY could increase the expressions of PGC-1a (0.77 ± 0.06), NRF-1 (0.70 ± 0.05), and TFAM (0.74 ± 0.07). These results suggested that CRY partly protected and promoted the mitochondrial biogenesis. Analysis on gene detection is shown in Figure 3(A), while analysis on protein detection is shown in Figure 3(B). Photos of protein expressions are shown in Figure 3(C). Effects of ADR on oxidative stress-relative enzymes The content of NO in myocardium of rat was normal in the NC group (49.67 ± 8.84 mmol/g Pro). Due to the toxicity on myocardium of rat, the content of NO in the ADR group was markedly increased (108.44 ± 10.80 mmol/g Pro). A remarkable augmentation of NO was detected after the treatment of CRY (82.51 ± 11.33 mmol/g Pro), despite higher than normal. The activity of iNOS was hardly detected in the NC group (9.50 ± 3.82 nmol/min/g Pro), while raised substantially in the ADR group (190.35 ± 25.02 nmol/min/g Pro) due to the toxicity. A remarkable augmentation of iNOS was detected after the treatment of CRY (133.17 ± 16.10 nmol/min/g Pro). No obvious changes on the activity of cNOS in myocardium
Figure 1. (A) The electron transport chain activities (complexes I–IV) in mitochondria in NC, ADR, and ADR+CRY groups. (B) The ATP generation in mitochondria in NC, ADR, and ADR+CRY groups. Data are shown as mean ± SD (n ¼ 6). Significant differences are defined as p50.05 (#ADR versus ADR+CRY) and p50.01 (**NC versus ADR).
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Figure 2. (A) The mitochondrial membrane potential in NC, ADR, and ADR+CRY groups. (B) The level of superoxide anion free radical in mitochondria in NC, ADR, and ADR+CRY groups. Data are shown as mean ± SD (n ¼ 6). Significant differences are defined as p50.05 (#ADR versus ADR+CRY) and p50.01 (**NC versus ADR).
Figure 3. (A) Analysis on the expressions of mitochondrial biogenesis relative factors PGC-1a, NRF-1, and TFAM at gene level. (B) Analysis on the expressions of mitochondrial biogenesis relative factors PGC-1a, NRF-1, and TFAM at protein level. (C) The protein expressions of mitochondrial biogenesis relative factors PGC-1a, NRF-1, and TFAM. Lane (1) NC group; (2) ADR group; (3) ADR+CRY group. Data are shown as mean ± SD (n ¼ 6). Significant differences are defined as p50.05 (*NC versus ADR; #ADR versus ADR+CRY) and p50.01 (**NC versus ADR; ##ADR versus ADR+CRY).
were found among NC (267.69 ± 19.72 nmol/min/g Pro), ADR (258.47 ± 16.39 nmol/min/g Pro), and ADR+CRY (266.01 ± 19.21 nmol/min/g Pro) groups. Compared with the NC group (181.88 ± 13.69 U/min/mg Pro), the activity of GSH-PX was greatly decreased in the ADR group (91.71 ± 9.78 U/min/mg Pro) due to the mitochondrial injury. It was significantly increased after the treatment of CRY (127.56 ± 16.09 U/min/mg Pro). Data are shown in Figure 4.
Discussion ADR is widely used as the first-line drug for the treatment of many cancers. The most serious adverse effect of ADR is
cardiomyopathy. It leads to congestive heart failure, depending on its cumulative dose through mitochondria (Ichikawa et al., 2014). Oxidative stress and mitochondrial dysfunction are main causes of cardiac dysfunction and cardiomyopathy (Faulk et al., 2013). CRY is extracted from the root of a medical herb called Salvia miltiorrhiza with the function of cardiovascular protection. We detected the mitochondrial protection of CRY against ADR-induced dysfunction in rat cardiomyocytes. Generation of ATP is the primary function of mitochondria. Four membrane-bound complexes (I, II, III, and IV) embedded in the inner membrane have been identified as
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DOI: 10.3109/13880209.2015.1029052
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Figure 4. The content of NO, iNOS, cNOS, and GSH-PX in mitochondria in NC, ADR, and ADR+CRY groups. Data are shown as mean ± SD (n ¼ 6). Significant differences are defined as p50.05 (#ADR versus ADR+CRY) and p50.01 (**NC versus ADR).
critical factors in ATP production (Sun et al., 2013). High activities will promote the ATP generation while suppressive activities will inhibit the process. This regulation plays an important role in cardiac myocytes (Sag et al., 2013). Due to the toxicity of ADR on mitochondria, the activities (complexes I–IV) of ETC were all markedly reduced, resulting in the suppressed ATP generation. After the treatment of CRY, a remarkable augmentation of the activities of the complexes I, III, and IV on ETC were detected. CRY promoted the activities of complexes I–IV except complex II. Complex II, also named as succinate dehydrogenase, is not involved in the protection of CRY on mitochondria. In accordance with the protection on complexes, the ATP generation was significantly increased. These results indicated CRY could promote the ATP generation by protecting the mitochondrial complexes I, III, and IV. The abnormal MMP plays a key role in cardiomyopathy (Kadenbach et al., 2011). Due to the toxicity on mitochondria, the MMP decreased significantly in the ADR group. It increased markedly after the treatment of CRY, despite lower than normal. The result indicated the partly repair of mitochondrial membrane by CRY. Changes in MMP-controlled matrix remodeling lead to increased superoxide anion free radical (Herlein et al., 2011). Matrix remodeling to the condensed state results in crystal unfolding and exposes cytochrome c to the intermembrane space facilitating superoxide anion free radical increased during the injury on mitochondria (Gupta et al., 2009; Na¨pa¨nkangas et al., 2012). Excessive generation of superoxide anion free radical amounts can break the balance in cellular reduction– oxidation (redox) and disrupt normal cellular functions. The redox post-translational modification regulates superoxide
anion free radical in the mitochondria and is strictly linked to mediating the physiological effects of mitochondrial functions. Due to the damage on mitochondria, the superoxide anion free radical was obviously increased in the ADR group. After the treatment of CRY, it was down-regulated significantly despite higher than normal. Inhibition of superoxide anion free radical release could reduce the damage to the mitochondria. Mitochondrial biogenesis is the process of forming new mitochondria in the cell. It is activated by cellular stress, environmental stimuli, and injury (Garesse & Vallejo, 2001; Poulose & Raju, 2014). PGC-1a, NRF-1, and TFAM are three most important factors in regulating the mitochondrial biogenesis. PGC-1a is an important regulator in mitochondrial biogenesis by interacting with and regulating the activities of cAMP response element-binding protein (CREB) and nuclear respiratory factors (NRFs) (Chowanadisai et al., 2010). NRF-1 activates the expression of some key metabolic genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication (Scarpulla et al., 2002). TFAM bends mitochondrial promoter DNA to aid transcription of the mitochondrial genome, regulating the mitochondrial genome copy number (Campbell et al., 2012). Our research found the expressions of these genes were markedly reduced due to the damage on mitochondria in the ADR group. This dysfunction caused by CRY significantly increased the expressions of these genes in the ADR + CRY group, suggesting the partly repair of the function of mitochondria. Mitochondria are the main source of reactive oxygen species (ROS) production in the cell (Davidson, 2010; Tang et al., 2011). Nitric oxide (NO) is an important cellular
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signaling indicator for the ROS production, biosynthesized endogenously from L-arginine, oxygen, and NADPH by nitric oxide synthases (NOSs) (Smith et al., 2005). NOSs are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine (Yada et al., 2007). After injury on mitochondria, NO was markedly increased while a remarkable augmentation was detected after the treatment of CRY. It was the same with the iNOS. No obvious change was detected on the activity of cNOS. These results indicated that it was iNOS rather than cNOS to be responsible for the increased level of NO. GSH-PX protects the organism from oxidative damage by reducing lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. Due to the mitochondrial injury, the activity of GSHPX was markedly decreased in the ADR group. The activity was significantly increased after the treatment of CRY. These results indicate that CRY protects and provides a normal vascular function and environment following injury by downregulation of ROS.
Conclusions In summary, CRY protects the energy production by increasing the complexes activities in ETC and the ATP generation. CRY also increases the MMP and inhibits the superoxide anion free radicals. The expressions of mitochondrial biogenesis-relative genes can be up-regulated by CRY. Remarkable augmentations of NO, iNOS, and increased activity of GSH-PX are detected after the treatment of CRY. However, these structural and functional protections are partly recovery. Our researches suggest that CRY could be a potential drug to prevent the cardiotoxicity of ADR. The pharmacokinetics, toxicological, dosage, and side effects of CRY will be further studied. This research is only performed on rats. There is still a long way before clinical application.
Declaration of interest The authors report that they have no conflicts of interest. This research was supported by the Research Program of Wuwei Tumor Hospital and Chinese PLA General Hospital.
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Cryptotanshinone protects against adriamycin-induced mitochondrial dysfunction in cardiomyocytes a
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Yanshan Zhang , Liang Chen , Fan Li , Huijuan Wang , Yunyi Yao , Jiamei Shu & Ming-Zhong c
Ying a
Department of Tumor Surgery, Wuwei Tumor Hospital, Wuwei, Gansu PR China,
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Department of Paediatrics, Changhai Hospital, Second Military Medical University, Shanghai, PR China, c
International Medical Center, Chinese PLA General Hospital, Beijing PR China,
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Department of Tumor Chemotherapy, Wuwei Tumor Hospital, Wuwei, Gansu, PR China,
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Research Center for Biochemistry and Molecular Biology, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu, PR China, and f
Department of Cardiology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu PR China Published online: 05 May 2015.
To cite this article: Yanshan Zhang, Liang Chen, Fan Li, Huijuan Wang, Yunyi Yao, Jiamei Shu & Ming-Zhong Ying (2015): Cryptotanshinone protects against adriamycin-induced mitochondrial dysfunction in cardiomyocytes, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1029052
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http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–6 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1029052
ORIGINAL ARTICLE
Cryptotanshinone protects against adriamycin-induced mitochondrial dysfunction in cardiomyocytes Yanshan Zhang1*, Liang Chen2*, Fan Li3, Huijuan Wang4, Yunyi Yao5, Jiamei Shu6, and Ming-Zhong Ying3 Department of Tumor Surgery, Wuwei Tumor Hospital, Wuwei, Gansu, PR China, 2Department of Paediatrics, Changhai Hospital, Second Military Medical University, Shanghai, PR China, 3International Medical Center, Chinese PLA General Hospital, Beijing, PR China, 4Department of Tumor Chemotherapy, Wuwei Tumor Hospital, Wuwei, Gansu, PR China, 5Research Center for Biochemistry and Molecular Biology, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu, PR China, and 6Department of Cardiology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, PR China
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Abstract
Keywords
Context: The serious side effect of Adriamycin (ADR) is cardiomyopathy. Cryptotanshinone (CRY) is widely and safely used as antioxidant with MTD more than 5 mg/g in rats (p.o). Objective: The objective of this study is to study the protection effects of CRY against ADR-induced mitochondrial dysfunction in cardiomyocytes. Materials and methods: The chemical administration lasted for 20 days with an effective dose of CRY (p.o.) at 50 mg/kg in rats. Mitochondrial respiratory chain complex activities, ATP generation, mitochondrial membrane potential (MMP), superoxide anion free radical, oxidative stress-relative enzymes, and mitochondrial biogenesis-relative factors in normal control, ADR (i.p., 1.25 mg/kg), and ADR (i.p., 1.25 mg/kg) + CYP (p.o., 50 mg/kg) groups were detected. Results: 50 mg/kg CRY significantly promoted the energy production of ATP (16.99 ± 2.38 nmol/g Pro) (Pro: Protein) by increasing the complexes activities except II (p40.05). After the treatment of CRY, the suppressed MMP was increased while superoxide anion free radical (0.57 ± 0.07/mg Pro) was inhibited markedly. Mitochondrial biogenesis-relative factors PGC-1a, NRF-1, and TFAM were also promoted. Remarkable augmentations of NO, inducible nitric oxide synthase (iNOS), and increased activity of GSH-PX (p50.05) were also detected after the treatment of CRY, while no obvious changes on the activity of nitric oxide synthase (cNOS; p40.05) were observed. Discussion and conclusion: These results suggest that CRY protects against ADR-induced mitochondrial dysfunction in cardiomyocytes. It could be an ideal potential drug of cardioprotection.
ATP generation, cardiovascular protection, mitochondrial biogenesis, mitochondrial complex activity, mitochondrial membrane potential, mitochondrial respiratory chain, oxidative stress, superoxide anion free radical
Introduction Adriamycin (ADR), also known as doxorubicin, is an anthracycline antibiotic drug for cancer chemotherapy by intercalating DNA of cancer cells. It is widely used as the first-line drug in the treatments of hematological malignancies, types of carcinoma, and soft tissue sarcomas. The most serious adverse effect is cardiomyopathy, leading to congestive heart failure (Octavia et al., 2012). Few effective drugs can be used to prevent the cardiotoxicity of ADR. Cryptotanshinone (CRY) is one of the most abundant constituents of the root of Salvia miltiorrhiza Bunge (Lamiaceae). It is widely used as a medical herb in Asian and European countries for the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the major bioactive compounds of Salvia miltiorrhiza roots including the *These authors contributed equally to this work. Correspondence: Prof. Ming-Zhong Ying, International Medical Center, Chinese PLA General Hospital, Beijing 100853, PR China. E-mail: [email protected]
History Received 12 August 2014 Revised 3 March 2015 Accepted 09 March 2015 Published online 10 April 2015
cryptotanshinone, are diterpene quinone, promoting the cardiovascular protection (Han et al., 2008). Unlike tanshinone I and tanshinone IIA, fewer studies of cryptotanshinone have been focused on the pharmacological effects including the cardiovascular protection. Research indicates ADRinduced cardiotoxicity via mitochondria (Carvalho et al., 2014; Finsterer & Ohnsorge, 2013). Given that CRY can adjust the oxidative stress (Chen et al., 2014), which is caused by mitochondria, this research mainly focused on the protection on cardiomyocytes via mitochondria instead of other organelles. Our previous research indicates that CRY is a safe chemical with no cytotoxicity or oncogenicity. The ED50 of CRY is 10 mg/kg in rats for cardioprotection according to our previous research. The effect is dosedependent. The maximum tolerated dose (MTD) of CRY is even more than 5 mg/g (p.o) in rats. Our former study tested the dose at 10, 20, 50, 100 mg/kg and indicated 50 and 100 mg/kg were the most effective doses for cardioprotection. No obvious differences were found between 50 and 100 mg/kg. The purpose of this study was to examine whether
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mitochondrial dysfunction could be relieved by CRY and the cardiovascular protection of CRY against ADR-induced mitochondrial dysfunction in rat cardiomyocytes.
Materials and methods
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Animal model generation and drug treatment Adult male Wistar rats weighing 300 ± 50 g (Beijing HFK Bioscience Co., LTD, Beijing, China) were assigned into three groups randomly, six rats each group: the normal control (NC) group, the ADR group, and the ADR + CRY group. Rats in the NC group were treated with 0.9% normal saline (i.p.) and 0.5% sodium carboxymethylcellulose (CMC-Na, p.o.) for a total of 20 d. Rats in the ADR group were treated with 0.9% normal saline (i.p.) and 0.5% CMC-Na (p.o.) for a total of 20 d. On the 9th day, rats were treated with 1.25 mg/kg ADR (i.p.) every 2 d, six times for a total of 12 d (2 d 6 doses). Our previous research found that 50 mg/kg CRY (p.o.) was an effective dose for cardioprotection in rats. Rats in the ADR+CRY group were treated with normal saline (i.p.) and CRY (dissolved in 0.5% CMC-Na, p.o., 50 mg/kg) for a total of 20 d. On the 9th day, rats were treated with 1.25 mg/kg of ADR (i.p.) every 2 d, six times for a total of 12 d (2 d 6 doses). Rats were sacrificed and myocardium was isolated on the 21st day. Mitochondria were purified by Percoll density gradient centrifugation according to a published paper (Morin et al., 2002). In accordance with the published guidelines of the China Council on Animal Care, this research was approved by the Medical Ethics Committee of Chinese PLA General Hospital and Wuwei Tumor Hospital. Determination of mitochondrial respiratory chain complexes activities and ATP generation The activities of mitochondrial respiratory chain complexes, complex I (NADH dehydrogenase, kit Cat.: A089-1), complex II (succinate dehydrogenase, kit Cat.: A089-2), complex III (ubiquinol cytochrome c reductase, kit Cat.: A089-3), and complex IV (cytochrome c oxidase, kit Cat.: A089-4) were analyzed using ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the instructions of the manufacturer. Briefly, blank wells, standard wells, and testing sample wells were set separately. Sample dilution (40 ll) was added into testing sample wells, then 10 ll testing sample was added (final dilution is 1/5), mixed gently, incubated at 37 C for 30 min, and then washed for five times. HRP-conjugate reagent (50 ml) was added to each well except blank well, incubated at 37 C for 30 min, and then washed. Chromogen solution A (50 ll) and chromogen solution B (50 ll) were added into each well for 15 min at 37 C and then stop solution (50 ll) was added into each well to stop the reaction for 15 min, waiting for the blue to change yellow. Blank wells were adjusted as 0. The absorbance in each well was recorded (k ¼ 450 nm). The value in the NC group was set as 1 while other relative values were calculated by comparison. The adenosine triphosphate (ATP) concentration in the mitochondria was measured using reverse-phase highpressure liquid chromatography (Agilent Technologies, Palo Alto, CA) following the protocol reported previously (Chen et al., 2005).
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Determination of mitochondrial membrane potential and superoxide anion free radical Mitochondrial membrane potential (MMP) was detected using 5,50 ,6,60 -Tetrachloro-1,10 ,3,30 -tetraethyl-imidacarbocyanine iodide (JC-1) staining kit (C2006, Beyotime Institute of Biotechnology, Shanghai, China). About 30 mg mitochondria sample (100 mL) was incubated with 900 mL JC-1 staining buffer. The fluorescence intensity was detected in the fluorescence spectrophotometer (F-7000, Hitachi, Tokyo, Japan) at 37 C for 20 min. The ratio of aggregates (k ¼ 590 nm) to monomer (k ¼ 530 nm) was calculated as an indicator of MMP. The value in the NC group was set as 1 while other relative values were calculated by comparison. The superoxide anion free radical was detected using a kit according to the instruction of the manufacturer (Nanjing Jiancheng Bioengineering Institute, China. kit Cat.: A052). Determination of mitochondrial biogenesis-relative factors The total RNA was isolated from myocardium in NC, ADR, and ADR + CRY groups and was reverse transcripted into cDNA as the templates for the Real-time qPCR detection (ABI7500, Applied Biosystems, Waltham, MA). Oligonucleotide primers for mitochondrial biogenesis-relative genes were peroxisome proliferator-activated receptor-g-coactivator 1-a (PGC-1a): up: 50 -TATTCCAGGTCAAGATCAA GGTCC-30 , down: 50 -CTTTCGTGCTCATTGGCTTCATAG30 , 185 bp; nuclear respiratory factor 1 (NRF-1): up: 50 -CAGA TAGTCCTGTCTGGGGAAACC-30 , down: 50 -CCGCCATAA TGAATCCCTTTCCAA-30 , 169 bp, mitochondrial transcription factor A (TFAM): up: 50 -CCATGGACTTCTGCCCAC TGAAT-30 , down: 50 -CTTCACAAACCCGCACGAAACTG30 , 204 bp, (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China). Total protein extracted (30 mg) from in NC, ADR, and ADR + CRY groups was boiled with 4 loading buffer at 100 C for 5 min and then injected to 12.5% SDS-PAGE. After electrophoresis, it was transferred to the PVDF membrane (Invitrogen, Waltham, MA), blocked in 5% fat-free milk for 1 h and incubated overnight at 4 C with anti-PGC-1 (K-15): (goat polyclonal IgG, sc-5816, 91 kDa, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, 1:600), anti-NRF-1 (rabbit monoclonal IgG, ab175932, 54 kDa, Abcam, Cambridge, MA, 1:1000), and anti-TFAM (rabbit polyclonal IgG, Catalog# 3885-100, 27 kDa, BioVision, Inc., Milpitas, CA, 1:800) and then secondary antibody for 1 h at room temperature. b-Actin was used as the native control. The value (target/b-actin) in the NC group was set as 1 while the relative data were calculated by comparing with the NC group. Determination of oxidative stress-relative enzymes Myocardium of rats in three groups were isolated and used for the detection of activities of oxidative stress-relative enzymes nitric oxide (NO) (A013-2), constitutive nitric oxide synthase (cNOS), and inducible nitric oxide synthase (iNOS) (A014-1), glutathione peroxidase (GSH-PX) (A005). Kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Chemical colorimetry was used for the detection in spectrophotometer.
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Statistical analysis All data were expressed as mean ± SD and analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL). The results were evaluated by one-way ANOVA. Statistical significances were defined as p50.05(*) and p50.01(**).
Results
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Effects of ADR on mitochondrial respiratory chain complexes activities and ATP generation Due to the toxicity of ADR on myocardium of rat, the activities of mitochondrial complexes I (46.68 ± 4.73), II (60.03 ± 7.51), III (44.30 ± 7.46), and IV (49.27 ± 5.02) in the ADR group were all greatly inhibited. Except complex II (67.31 ± 7.10, p40.05), remarkable augmentations of the activities of complexes I (65.14 ± 9.21), III (58.73 ± 4.48), and IV (63.29 ± 7.04) were detected after the treatment of CRY, despite lower than normal. Data are shown in Figure 1(A). The activities of complexes regulate the ATP generation. Low activities of mitochondrial complexes will result in the suppressed ATP generation. The ATP generation was normal in the NC group (36.48 ± 5.79 nmol/g) Prowhile greatly inhibited in the ADR group (8.90 ± 2.36 nmol/g Pro). After the treatment of CRY, the ATP generation (16.99 ± 2.38 nmol/g Pro) was markedly promoted, despite lower than normal. These results suggested the functional recovery of ETC by CRY. Taken together, these results indicated that CRY promoted the ATP generation through the activity recoveries of complexes I, III, and IV. Data are shown in Figure 1(B). Effects of ADR on the mitochondrial membrane potential and superoxide anion free radical CRY could promote the structural repair and functional recovery on mitochondrial membrane. Due to the damage of ADR on mitochondria, the MMP was markedly decreased in the ADR group (0.61 ± 0.08). After the treatment of CRY, it was significantly increased (0.78 ± 0.07), despite lower than normal. Data are shown in Figure 2(A). As an indicator of oxidative stress, the release of superoxide anion free radical
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was markedly increased in the ADR group (0.75 ± 0.08/mg Pro) compared with the NC group (0.26 ± 0.06/mg Pro). After the treatment of CRY, it was significantly inhibited (0.57 ± 0.07/mg Pro). The damage on mitochondria caused by oxidative stress could also be weakened after the inhibition. Data are shown in Figure 2(B).
Effects of ADR on mitochondrial biogenesis-relative factors The function of mitochondrial biogenesis was weakened because of the toxicity of ADR on mitochondria. On the gene level, ADR down-regulated the expressions of mitochondrial biogenesis-relative factors PGC-1a (0.57 ± 0.06), NRF-1 (0.54 ± 0.09), and TFAM (0.60 ± 0.10). The expressions of PGC-1a (0.72 ± 0.07), NRF-1 (0.75 ± 0.08), and TFAM (0.80 ± 0.07) were increased significantly after the treatment of CRY, despite lower than the NC group. On the protein level, PGC-1a (0.58 ± 0.07), NRF-1 (0.41 ± 0.07), and TFAM (0.53 ± 0.08) were also down-regulated in the ADR group. CRY could increase the expressions of PGC-1a (0.77 ± 0.06), NRF-1 (0.70 ± 0.05), and TFAM (0.74 ± 0.07). These results suggested that CRY partly protected and promoted the mitochondrial biogenesis. Analysis on gene detection is shown in Figure 3(A), while analysis on protein detection is shown in Figure 3(B). Photos of protein expressions are shown in Figure 3(C). Effects of ADR on oxidative stress-relative enzymes The content of NO in myocardium of rat was normal in the NC group (49.67 ± 8.84 mmol/g Pro). Due to the toxicity on myocardium of rat, the content of NO in the ADR group was markedly increased (108.44 ± 10.80 mmol/g Pro). A remarkable augmentation of NO was detected after the treatment of CRY (82.51 ± 11.33 mmol/g Pro), despite higher than normal. The activity of iNOS was hardly detected in the NC group (9.50 ± 3.82 nmol/min/g Pro), while raised substantially in the ADR group (190.35 ± 25.02 nmol/min/g Pro) due to the toxicity. A remarkable augmentation of iNOS was detected after the treatment of CRY (133.17 ± 16.10 nmol/min/g Pro). No obvious changes on the activity of cNOS in myocardium
Figure 1. (A) The electron transport chain activities (complexes I–IV) in mitochondria in NC, ADR, and ADR+CRY groups. (B) The ATP generation in mitochondria in NC, ADR, and ADR+CRY groups. Data are shown as mean ± SD (n ¼ 6). Significant differences are defined as p50.05 (#ADR versus ADR+CRY) and p50.01 (**NC versus ADR).
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Figure 2. (A) The mitochondrial membrane potential in NC, ADR, and ADR+CRY groups. (B) The level of superoxide anion free radical in mitochondria in NC, ADR, and ADR+CRY groups. Data are shown as mean ± SD (n ¼ 6). Significant differences are defined as p50.05 (#ADR versus ADR+CRY) and p50.01 (**NC versus ADR).
Figure 3. (A) Analysis on the expressions of mitochondrial biogenesis relative factors PGC-1a, NRF-1, and TFAM at gene level. (B) Analysis on the expressions of mitochondrial biogenesis relative factors PGC-1a, NRF-1, and TFAM at protein level. (C) The protein expressions of mitochondrial biogenesis relative factors PGC-1a, NRF-1, and TFAM. Lane (1) NC group; (2) ADR group; (3) ADR+CRY group. Data are shown as mean ± SD (n ¼ 6). Significant differences are defined as p50.05 (*NC versus ADR; #ADR versus ADR+CRY) and p50.01 (**NC versus ADR; ##ADR versus ADR+CRY).
were found among NC (267.69 ± 19.72 nmol/min/g Pro), ADR (258.47 ± 16.39 nmol/min/g Pro), and ADR+CRY (266.01 ± 19.21 nmol/min/g Pro) groups. Compared with the NC group (181.88 ± 13.69 U/min/mg Pro), the activity of GSH-PX was greatly decreased in the ADR group (91.71 ± 9.78 U/min/mg Pro) due to the mitochondrial injury. It was significantly increased after the treatment of CRY (127.56 ± 16.09 U/min/mg Pro). Data are shown in Figure 4.
Discussion ADR is widely used as the first-line drug for the treatment of many cancers. The most serious adverse effect of ADR is
cardiomyopathy. It leads to congestive heart failure, depending on its cumulative dose through mitochondria (Ichikawa et al., 2014). Oxidative stress and mitochondrial dysfunction are main causes of cardiac dysfunction and cardiomyopathy (Faulk et al., 2013). CRY is extracted from the root of a medical herb called Salvia miltiorrhiza with the function of cardiovascular protection. We detected the mitochondrial protection of CRY against ADR-induced dysfunction in rat cardiomyocytes. Generation of ATP is the primary function of mitochondria. Four membrane-bound complexes (I, II, III, and IV) embedded in the inner membrane have been identified as
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Figure 4. The content of NO, iNOS, cNOS, and GSH-PX in mitochondria in NC, ADR, and ADR+CRY groups. Data are shown as mean ± SD (n ¼ 6). Significant differences are defined as p50.05 (#ADR versus ADR+CRY) and p50.01 (**NC versus ADR).
critical factors in ATP production (Sun et al., 2013). High activities will promote the ATP generation while suppressive activities will inhibit the process. This regulation plays an important role in cardiac myocytes (Sag et al., 2013). Due to the toxicity of ADR on mitochondria, the activities (complexes I–IV) of ETC were all markedly reduced, resulting in the suppressed ATP generation. After the treatment of CRY, a remarkable augmentation of the activities of the complexes I, III, and IV on ETC were detected. CRY promoted the activities of complexes I–IV except complex II. Complex II, also named as succinate dehydrogenase, is not involved in the protection of CRY on mitochondria. In accordance with the protection on complexes, the ATP generation was significantly increased. These results indicated CRY could promote the ATP generation by protecting the mitochondrial complexes I, III, and IV. The abnormal MMP plays a key role in cardiomyopathy (Kadenbach et al., 2011). Due to the toxicity on mitochondria, the MMP decreased significantly in the ADR group. It increased markedly after the treatment of CRY, despite lower than normal. The result indicated the partly repair of mitochondrial membrane by CRY. Changes in MMP-controlled matrix remodeling lead to increased superoxide anion free radical (Herlein et al., 2011). Matrix remodeling to the condensed state results in crystal unfolding and exposes cytochrome c to the intermembrane space facilitating superoxide anion free radical increased during the injury on mitochondria (Gupta et al., 2009; Na¨pa¨nkangas et al., 2012). Excessive generation of superoxide anion free radical amounts can break the balance in cellular reduction– oxidation (redox) and disrupt normal cellular functions. The redox post-translational modification regulates superoxide
anion free radical in the mitochondria and is strictly linked to mediating the physiological effects of mitochondrial functions. Due to the damage on mitochondria, the superoxide anion free radical was obviously increased in the ADR group. After the treatment of CRY, it was down-regulated significantly despite higher than normal. Inhibition of superoxide anion free radical release could reduce the damage to the mitochondria. Mitochondrial biogenesis is the process of forming new mitochondria in the cell. It is activated by cellular stress, environmental stimuli, and injury (Garesse & Vallejo, 2001; Poulose & Raju, 2014). PGC-1a, NRF-1, and TFAM are three most important factors in regulating the mitochondrial biogenesis. PGC-1a is an important regulator in mitochondrial biogenesis by interacting with and regulating the activities of cAMP response element-binding protein (CREB) and nuclear respiratory factors (NRFs) (Chowanadisai et al., 2010). NRF-1 activates the expression of some key metabolic genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication (Scarpulla et al., 2002). TFAM bends mitochondrial promoter DNA to aid transcription of the mitochondrial genome, regulating the mitochondrial genome copy number (Campbell et al., 2012). Our research found the expressions of these genes were markedly reduced due to the damage on mitochondria in the ADR group. This dysfunction caused by CRY significantly increased the expressions of these genes in the ADR + CRY group, suggesting the partly repair of the function of mitochondria. Mitochondria are the main source of reactive oxygen species (ROS) production in the cell (Davidson, 2010; Tang et al., 2011). Nitric oxide (NO) is an important cellular
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signaling indicator for the ROS production, biosynthesized endogenously from L-arginine, oxygen, and NADPH by nitric oxide synthases (NOSs) (Smith et al., 2005). NOSs are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine (Yada et al., 2007). After injury on mitochondria, NO was markedly increased while a remarkable augmentation was detected after the treatment of CRY. It was the same with the iNOS. No obvious change was detected on the activity of cNOS. These results indicated that it was iNOS rather than cNOS to be responsible for the increased level of NO. GSH-PX protects the organism from oxidative damage by reducing lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. Due to the mitochondrial injury, the activity of GSHPX was markedly decreased in the ADR group. The activity was significantly increased after the treatment of CRY. These results indicate that CRY protects and provides a normal vascular function and environment following injury by downregulation of ROS.
Conclusions In summary, CRY protects the energy production by increasing the complexes activities in ETC and the ATP generation. CRY also increases the MMP and inhibits the superoxide anion free radicals. The expressions of mitochondrial biogenesis-relative genes can be up-regulated by CRY. Remarkable augmentations of NO, iNOS, and increased activity of GSH-PX are detected after the treatment of CRY. However, these structural and functional protections are partly recovery. Our researches suggest that CRY could be a potential drug to prevent the cardiotoxicity of ADR. The pharmacokinetics, toxicological, dosage, and side effects of CRY will be further studied. This research is only performed on rats. There is still a long way before clinical application.
Declaration of interest The authors report that they have no conflicts of interest. This research was supported by the Research Program of Wuwei Tumor Hospital and Chinese PLA General Hospital.
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