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Polysaccharides from the peels of Citrus aurantifolia induce apoptosis in transplanted H22 cells in mice
Accepted Manuscript Title: Polysaccharides from the peels of Citrus aurantifolia induce apoptosis in transplanted H22 cells in mice Authors: Yana Zhao, Hongyan Sun, Ling Ma, Anjun Liu PII: DOI: Reference:
S0141-8130(16)32520-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.149 BIOMAC 7315
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19-11-2016 14-3-2017 19-3-2017
Please cite this article as: Yana Zhao, Hongyan Sun, Ling Ma, Anjun Liu, Polysaccharides from the peels of Citrus aurantifolia induce apoptosis in transplanted H22 cells in mice, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.149 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Polysaccharides from the peels of Citrus aurantifolia induce apoptosis in transplanted H22 cells in mice Yana Zhao, Hongyan Sun, Ling Ma, Anjun Liu*
Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
* Corresponding author.
Tel./fax: +86 22 60912390
E-mail address: [email protected]
Highlights:
An acidic polysaccharide (CAs) with the molecular weight of 7.94 × 106 Da, which mainly composed of rhamnos, arabinose, galactose, glucose, mannose and galacturonic acid, was obtained from Citrus aurantifolia peels.
CAs had capacity of repressing transplanted H22 cells growth in vivo and exhibiting few toxic effects on host animals.
CAs was able to improve the levels of tumor infiltrating CD8+ T lymphocytes, block cell cycle in S phase, trigger apoptosis-related proteins expression and induce transplanted H22 cells apoptosis in vivo.
Abstract
In this study, an acidic polysaccharide (CAs) was extracted and purified from the peels of Citrus aurantifolia by Sephadex G-150. HPGPC showed the molecular weight of CAs was about 7.94 × 106 Da. Ion chromatography (IC) analysis showed CAs was mainly composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glu), mannose (Man) and galacturonic acid 1
(GalA), with the molar ratio of 0.67: 7.67: 10.83: 3.83: 4.00: 1.00. 1H and 13C NMR spectra of CAs also identified the presence of five kinds of monosaccharides and galacturonic acid. Moreover, the antitumor activity of CAs was evaluated in mice transplanted H22 hepatoma cells. It was shown that CAs dose-dependently suppressed tumor cells growth with few toxic effects on host. Further investigations revealed that CAs increased the levels of tumor infiltrating CD8+ T lymphocytes, blocked tumor cell cycle in S phase, down-regulated anti-apoptotic protein Bcl-xL and Mcl-1 expression, and led to the activation of caspase 3. These results suggested that CAs had capacity of inducing tumor cells apoptosis in vivo, and it supported considering CAs as an adjuvant reagent in hepatocellular carcinoma treatment.
Keywords : Citrus aurantifolia peels; Polysaccharides; Hepatocellular carcinoma.
1 Introduction
Primary liver cancer is the most common malignant tumor overall and the second most common cause of cancer mortality worldwide. Hepatocellular carcinoma (HCC) accounts for up to 90% of all primary hepatic malignancies and represents a major health problem. Surgical resection is generally regarded as the most effective strategy for treating HCC. However, more than 80% of HCC patients are not eligible to liver transplantation, surgical resection, or liver regional therapy due to advanced disease or poor liver function [1, 2]. Therefore, systemic chemotherapy is beneficial for the majority of patients. Although several compounds such as 5-fluorouracil (5-FU), sorafenib, and cisplatin have displayed a potential antitumor effect and have been already approved for HCC treatment, their prognosis is often
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impaired by the drug resistance of HCC cells and there are a series of toxicity impacts on the normal cells and organs of patients [3]. Thus, it is urgent to search for new chemicals capable of provoking tumor cells death but exhibiting low adverse effects on the patients. Recently, polysaccharides and polysaccharide-protein complexes from traditional Chinese plants have been used as potential antitumor therapeutic agents, and their obvious antitumor activity and relatively low toxicity have been constantly confirmed [4, 5].
Apoptosis is an evolutionarily conserved program for cell self-destruction in multicellular organism indispensable for many biological processes such as development, growth, and homeostasis. It could be characterized by molecular features including chromatin condensation, DNA fragmentation, membrane blebbing, cell shrinkage and formation of apoptotic body [6, 7]. To date, the extrinsic death receptor pathway and the intrinsic mitochondrial pathway are two main apoptosis pathways, and they could be triggered by various chemical, physical and biological signals, such as chemical reagents, free radicals, radiation, and virus infection et al [8, 9]. Currently, most of therapeutic reagents achieve the purpose of antitumor through improving host immune response, suppressing tumor cell growth, arresting cell cycle at a specific phase and thereby inducing cell apoptosis in vivo or vitro. For example, corn peptides could induce Hep G2 cells apoptosis through mitochondrial apoptosis pathway in vitro and enhance host immune response in H22 tumor-bearing mice [10]. Kurarinol induced HCC cell apoptosis through repressing cellular signal transducer and activator of transcription 3 (STAT3) in vitro and vivo [11]. Therefore, the induction of
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apoptosis has been verified as a promising strategy for the prevention and treatment of hepatocellular carcinoma.
Numbers of researches have showed that citrus peels are a promising phytochemical source with a great deal of functional components, such as phenolic, limonoids, flavonoids and polysaccharides. In some regions of the world, especially in East-Asia including China, citrus peels have been used for traditional herbal medicine as antioxidant, antibacterial, antiinflammatory and antitumor [12, 13]. Nobiletin is a fraction of Citrus flavonoid, which has been demonstrated anti-HCC activity both in vitro and vivo [14]. Polysaccharides from Korean Citrus hallabong peels inhibit angiogenesis and breast cancer cell migration in vitro [15]. It also reported that pectic polysaccharides from the peels of Korean Citrus Hallabong have obvious antitumor activity via stimulation of macrophages and NK cells [16]. Citrus aurantifolia is commonly called as key lime or bitter orange and belongs to the Rutaceae Family. C. aurantifolia peels are main by-product and are usually discarded in processing citrus. Nevertheless, the extracts from C. aurantifolia peels are an attractive source of biochemicals (e.g., flavonoids, pectin, essential oil, polysaccharides) with potential health benefits [17, 18]. However, there are few reports on the composition and biological activity of C. aurantifolia peels polysaccharides. In this work, an acidic polysaccharide was isolated and purified from the peels of C. aurantifolia. Moreover, antitumor activity and possible mechanism of the polysaccharide against transplanted H22 cells were also evaluated in vivo.
2 Materials and methods
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2.1 Materials and reagents
Citrus aurantifolia was obtained from Haikou City, China. D-fucose, D-rhamnose, Larabinose, D-xylose, D-mannose, D-glucose, D-galactose, D-fructose, D-gluconic acid, Dgalacturonic acid, T-series dextran standards, propidium iodine (PI) and RIPA lysis buffer were purchased from Solarbio Co. (Beijing, China). Sephadex G-150 and bovine albumin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bradford Protein Assay Kit was purchased from Nanjing Jiancheng Biotech. Co. (Jiangsu, China). Primary antibodies were purchased from Tianjin Sungene Biotech Co. (Tianjin, China). Secondary antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA). The other chemical regents were of analytical grade.
2.2 Extraction, separation and purification of polysaccharides
C. aurantifolia peels were separated and dried in an oven at 40 ℃ until constant weight, then crushed into powder (particle size: 0.6 mm). The powder was refluxed with petroleum ether for 2 h to remove some colored materials, grease and other low molecular compounds. Then the residue was dried in air and extracted with 0.3 M NaOH (ratio of solid to liquid = 1:10) for three times (2 h each time) at room temperature. The supernatant was collected and the value of pH was adjusted to 3.0 using 0.1 M HCl, following precipitated by 80% (v/v) anhydrous ethanol and incubated at 4 ℃ overnight to obtain crude polysaccharides by centrifugation (3500 × g, 20 min).
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The crude polysaccharides were deproteinated using n-butanol and chloroform (1:4, v/v) according to the method of Sevage [19]. Then the mixture was concentrated, dialyzed (Mw 3500) with tap water for 48 h and distilled water for 48 h and lyophilized. The gained polysaccharides were further purified by Sephadex G-150 chromatography (2.6 cm × 35 cm). The column was eluted with distilled water at 0.8 ml/min and the polysaccharide fractions were collected by detecting carbohydrate with phenol-sulfuric acid method [20]. An acidic polysaccharide fraction was obtained and designated as CAs, which was used for next investigation.
2.3 Physicochemical properties of CAs
2.3.1 Ultraviolet (UV) and infrared (IR) analysis
CAs was dissolved with distilled water and diluted to proper concentration, then scanned from 200 to 900 nm with a Genesys 10s UV-VIS spectrophotometer (Thermo Scientific, America). IR spectrum of CAs was detected with a Fourier transformed infrared spectrophotometer (Bruker Vector-220, German) in the wave range of 4000 to 400 cm-1 using the KBr-disk method.
2.3.2 Chemical components analysis
Total carbohydrate content in CAs was tested by phenol-sulfuric acid method with galactose as standard [20]. Protein content in CAs was measured according to coomassie brilliant blue method using BSA as standard [21]. Uronic acid content in CAs was quantified by sulfuric acid-carbazole method using galacturonic acid as reference [22].
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2.3.3 Homogeneity and molecular weight analysis
The homogeneity and molecular weight of CAs was determined by high-performance gel-permeation chromatography (HPGPC) (Agilent-1200) equipped with a Tsk-gel G4000PWxl column (7.8 mm × 300 mm, column temperature 30 ℃) and Refractive Index Detector (RID, detecting temperature 35 ℃). 20 ul of polysaccharide solution was eluted at a flow rate of 0.8 ml/min with distilled water. The standard curve was established with T-series dextran (T-10, 40, 70, 110, 500 and 2000) as standard, and the molecular weight of CAs was calculated according to the calibration curve.
2.3.4 Monosaccharide composition analysis
The monosaccharide composition of CAs was detected by ion chromatography (IC), performed on a Dionex ISC5000 chromatographic system (CA, USA) with an efficient anion exchange column of Dionex Carbopac PA20 column (150 mm × 3 mm) and a Dionex pulsed amperometric detector equipped with an Au electrode. The polysaccharide was hydrolyzed as described [23], with some modifications. Chiefly, polysaccharide (5 mg) was dissolved in 2 M trifluoroacetic acid (TFA) and hydrolyzed at 120 ℃ for 6 h. Then TFA was absolutely removed by adding methanol and N2 was used as the carrier gas. The dried hydrolysate was dissolved with deionized water. NaOH solution (10 mM) and sodium acetate solution (200 mM) were used as mobile phase with gradient elution at a flow rate of 0.5 ml/min. Fucose, rhamnose, arabinose, xylose, mannose, glucose, galactose, fructose, glucuronic acid and galacturonic acid were used as references.
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2.3.5 Nuclear magnetic resonance (NMR) analysis
The polysaccharide was exchanged three times with deuterium in D2O by lyophilizing. 1
H and 13C NMR spectra were recorded on a NMR spectrometer (Brucker DRX-500) at 600
MHz.
2.4 Animals and animal experiments
ICR mice (female, 8 weeks old, weighting 20 ± 2 g) were purchased from the Department of Experimental Animal, Academy of Military Medical Science, Beijing, China. All the mice were under barrier conditions in the Center of Experimental Animals at Tianjin University of Science and Technology. The mice were allowed free access to food and water and were kept constant room temperature of 24 ± 1 ℃, 50 ± 10% relative humidity with a 12/12 h light/dark cycle. All animal handing procedures were performed strictly followed the Principles of Laboratory Animal Care (NIH Publication No.86-23, revised 1985).
In an array of previous studies, the experimental doses of plant polysaccharides on mice were around 100 mg/kg [24, 25]. In addition, for some functional components in citrus peels, we observed the Citrus peels flavonoids on H22-bearing mice were 125 - 500 mg/kg [14], and Citrus unshiu peel extracts on mice renal cell carcinoma model were 30 mg/kg [26]. Therefore, we selected two doses of CAs (50 and 250 mg/kg) as the low and the high dose in this study. Next, the mice were randomly divided into groups with ten mice in each group and treated as described in the text. Blank group and model group were both applied physiological saline solution orally throughout the experimental period. CAs groups were given CAs
8
solution with 50 or 250 mg/kg body weight every day. Two weeks later, all the mice in model group and CAs groups were subcutaneously injected mouse H22 ascitic hepatoma cells (obtained from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) in the right thigh of the lower limb with 0.2 ml (5 × 106 cells/ml). Then all the mice received the treatment for three weeks successively. At the end of the experiment, each group mice were weighed. The tumor volume was measured with a caliper and calculated according to following formula: Tumor volume (cm3) = LW2/2，wherein L and W stand for tumor length and width, respectively [27]. Then all the mice were sacrificed by cervical dislocation. The tumor tissues were dissected out and immediately weighed. The tumor inhibitory rate (TIR) was calculated by the formula: Tumor inhibitory rate (%) = (W1 – W2)/W1 × 100%. Where, W1 and W2 stand for the average tumor weight of model group and CAs groups mice, respectively [28].
2.5 Tumor infiltrating lymphocytes (TILs) analysis
Tumor tissues were collected under aseptic conditions from sacrificed mice and smashed with scissors, then gently passed through sterilized steel mesh to obtain homogeneous tumor cells suspension. The cells were adjusted to 1 × 106 cells/ml and incubated with either CD3FITC, CD4-PE, CD8-FITC, or CD19-PE monoclonal antibody in dark for 30 min at 4 ℃, then washed with PBS to remove unbounded antibody. The percentages of TILs were measured by flow cytometry (BD FACSCalibur, USA), and analyzed by CellQuest Pro. software.
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2.6 Cell cycle analysis
Tumor cells suspension was obtained as described previously. The cells were washed with PBS and adjusted to 1 × 106 cells/ml, then fixed with cold 70% ethanol (stored at -20 ℃) overnight. After washing, intracellular RNA was removed by incubating samples with RNase A (0.1 mg/ml) at 37 ℃ for 30 min. Then they were stained with PI (50 μg/ml) in dark for 30 min at 37 ℃. Cell cycle was detected by flow cytometry (BD FACSCalibur, USA), and analyzed by ModFit LT software.
2.7 Histology analysis
Tumor tissues were fixed with 10% (v/v) buffered formalin, dehydrated through a graded series of ethanol, cleared in xylene and embedded in paraffin. Slices (5-6 μm thick) of tumor tissues were prepared from paraffin blocks and stained with hematoxylin-eosin (H&E). Histopathological changes were examined by microscopy.
2.8 Western Blot analysis
Tumor tissues were lysed in RIPA lysis buffer according to manufacture’s instruction. The supernatant was collected and centrifuged at 10000 g for 5 min. Concentrations of proteins were quantified using Bradford Protein Assay Kit. The supernatant proteins were mixed with 1 × reducing electrophoresis loading buffer and boiled for 5 min. Protein samples (50-70 μg) were separated by 12% SDS-PAGE and transferred to PVDF membrane at 15 V by semi-dry blotting. Membranes were blocked with 5% bovine albumin in TBST for 2 h at room temperature and incubated with primary antibody overnight at 4 ℃. Primary antibodies
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were β-actin antibody (1:10000), Bcl-xL antibody (1:1000), Mcl-1 antibody (1:1000), and caspase 3 antibody (1:1000) were diluted in blocking solution. Membranes were washed with TBST three times (5 min/wash), and further incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-mouse IgG 1:10000 or goat antirabbit IgG 1:5000) for 1.5 h at room temperature. Finally, protein bands were examined using chemiluminescence (ECL) reagents and exposed to X-ray photographic films in a darkroom. The bands were visualized and analyzed with Quantity One software.
2.9 Statistical analysis
All experiments were performed at least three times. Experimental data were presented as mean ± standard deviation. Significant differences were established through analysis of variance (ANOVA) and mean comparisons were achieved by Duncan’s multiple range test. Data analysis was evaluated using SPSS software (SPSS Inc., Chicago, IL, USA). *p < 0.05 was considered as significant, and **p < 0.01 was considered as very significant.
3 Results and discussion
3.1 Isolation and purification of polysaccharide
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Fig. 1. The elution curve of polysaccharide fractions on Sephadex G-150. OD value at 490 nm states the content of carbohydrate by phenol-sulfuric acid method. The main fraction was named CAs.
The crude polysaccharide was obtained from the dried C. aurantifolia peels powder, and the yield of dry material was about 2.89%. As shown in Fig. 1, the crude polysaccharide was further separated by Sephadex G-150 and two fractions were gained. The second fraction was excluded in the following structure characterization due to the presence of some colored materials and low carbohydrate content based on the phenol-sulfuric acid assay. Thus, main product was collected and named CAs.
3.2 Chemical characterization of CAs
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Fig. 2. Spectra analysis of CAs. (A) UV spectrum of CAs in wavenumber region between 200 and 900 nm. (B) IR spectrum of CAs in the wavenumber region between 4000 and 400 cm-1.
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Fig. 3. Characterization of CAs. (A) The molecular weight of CAs was determined by HPGPC analysis. (B) The monosaccharide composition of CAs was measured by IC analysis. (a) Standard samples (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, gluconic acid, galacturonic acid arranged according to the peak time); (b) CAs sample (rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid arranged according to the peak time).
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Fig. 4. NMR spectra of CAs. (A) 1H NMR spectrum; (B) 13C NMR spectrum.
The UV spectrum analysis (Fig. 2A) showed that CAs had a weak absorption at 280 nm, indicating the presence of protein in polysaccharide. The IR spectrum of CAs was showed in Fig. 2B, which revealed classical polysaccharide absorbance peaks. The broadly intense peak
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at 3416 cm-1 was the stretching vibration of hydroxyl groups, and weak band at 2925 cm-1 belonged to the saturated C-H stretching vibration. The band at 1628 cm-1 due to N-H (CONH-) variable angle vibration, suggesting the presence of binding protein in polysaccharide [8]. The band at 1416 cm-1 was ascribed to -COOH stretching vibration. The intense peak at 1050 cm-1 was assigned the C-O-C glycosidic linkage. The peak at 896 cm-1 was attributed to the presence of α-D-pyranoid glucose, and the peak at 777 cm-1 may suggest the presence of α-isomers of pyranose [29].
The carbohydrate content of CAs was about 80.33% based on the phenol-sulfuric acid method. The protein content was about 4.36% by the Coomassie brilliant blue method. The uronic acid content of CAs was about 10.56% based on the sulfuric acid-carbazole method. The results showed that a small quantity of polypeptides or protein combined with polysaccharide.
The HPGPC analysis (Fig. 3A) showed that CAs only had one main peak with the retention time of 7.931, indicating that it was a homogeneous polysaccharide and the average molecular weight was 7.94 × 106 Da calculated in accordance with the retention time of standard dextrans. The standard curve of molecular weight was Y= -0.4367X + 10.429, R2 = 0.9902 (Y = log Mw, X = Rt). Analysis of monosaccharide composition of CAs using IC (Fig. 3B) showed that it was composed of rhamnose, arabinose, galactose, glucose, mannose and galacturonic acid, with the molar ratio of 0.67: 7.67: 10.83: 3.83: 4.00: 1.00.
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The structural features of CAs were further verified by NMR spectra analysis (Fig. 4). The chemical shifts at 5.020 - 5.375 ppm in the 1H NMR spectrum could be assigned to the typical signals of anomeric protons. The four anomeric protons signals at 5.375 (5.332), 5.186, 5.114 (5.084), 5.020 indicated CAs was mainly composed of glucose, galactose, mannose and arabinose. The signal at 1.177 ppm proved the presence of methyl protons in rhamnose. In 13C NMR spectrum, the signal at 177.53 ppm showed the presence of carboxyl carbon, which supported the conclusion that CAs was consisted of galacturonic acid. The four major signals indicated that the polysaccharide was composed of four kinds of monosaccharides. Additionally, the signal at 16.71 ppm revealed the presence of rhamnose in CAs.
3.3 Effect of CAs on transplanted H22 cell growth
Table 1
The effects of CAs on body weight, number, and tumor inhibitory rate (TIR) of H22-bearing mice Group
Dose
Mice body weight (g)
Number of mice
TIR
(mg/kg)
Start
End
Start/end
(%)
Blank group
-
22.92 ± 1.01
31.91 ± 1.61**
10/ 10
Model group
-
22.22 ± 1.34
28.07 ± 1.38
10/ 9
CAs
50
22.81 ± 1.62
29.73 ± 1.37
10/ 10
29.28
CAs
250
22.00 ± 2.32
30.88 ± 1.65*
10/ 10
58.85
18
* p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
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Fig. 5. The antitumor effect of CAs on H22-bearing mice. (A) The tumor pictures of mice in model and CAs groups. (B) Effect of CAs on the tumor weight of mice. (C) Effect of CAs on the tumor volume of mice. Fig. 5(A) was taken by a digital camera (PowerShot SD 630, Canon), and the tumor size was measured using a caliper. ** p < 0.01 was very significantly different compared to model group.
As shown in Table 1, in the initial stage of experiment, there were no significant differences of body weight among all groups. However, at the end of the experiment period, the body weight in model group obviously declined compared to blank group (p < 0.01). This might be caused by the rapidly growing tumor tissues. Meanwhile, we observed that mice in model group had thin body, slow autonomic movement and sparse hair. In contrast, the average body weight in CAs groups were no significant differences compared to blank group, and it had a significant increase compared to model group (p < 0.05), indicating that CAs treatment exhibited few negative impacts on body weight of tumor-bearing mice. Additionally, mice in CAs groups kept a normal body weight, good appetite and shiny fur. To further assess inhibitory activity of CAs, the average tumor weight and tumor volume were noted. As shown in Fig. 5, the average tumor weight and tumor volume in model group were significantly higher than CAs groups (p < 0.01). It was also shown that antitumor effect of CAs at 250 mg/kg was superior to the dose of 50 mg/kg, suggesting that the beneficial effect was performed in a dose-dependent manner. Moreover, the tumor inhibition rate (TIR) was also calculated in this work. As the results showed in Table 1, the TIR attained 29.28% and 58.85% in two doses of CAs, respectively, which revealed CAs had capacity of repressing
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H22 solid tumor growth. Similarly, according to the research of G.H. Mao et al. [30], a watersoluble polysaccharide from Grifola frondosa significantly inhibited the growth of Heps cells in vivo and tumor inhibition rate attained 56.16%. Q. Zhu et al. [31] found that Laminaria japonica polysaccharide improved immunolomodulatory activity and decreased tumor weight in H22-bearing mice. Simultaneously, tumor inhibition rate increased in a dose-dependent manner and it reached up to 59.67%. In addition, other polysaccharides, such as from Gentiana scabra Bunge [32], and Chaenomeles speciose [33], also have been investigated S180-repressing activity in vivo, and the highest tumor inhibition rate could attain 65.76% and 44.9%, respectively. Tumor inhibition rates of these polysaccharides are similar with the antitumor activity of CAs, supporting the application of Citrus aurantifolia peels in hepatocellular carcinoma treatment.
5-FU, cisplatin and sorafenib are conventional chemotherapeutic drugs widely applied in clinical HCC treatment, which present obvious tumor-suppressing activity. The research showed that 5-FU had potential inhibitory impacts on the growth of Heps tumors, and the inhibitory rate reached 68.49% compared to negative group. However, a significant decrease in spleen and thymus relative weight was also observed in 5-FU group [34]. Consistently, it also has been reported that cisplatin showed stronger HCC-suppressing activity with a tumor inhibition rate of 79.4%. However, at the end of 10 days treatment, 2 out of 10 mice died in cisplatin group [28]. In addition, the most frequently reported the adverse events in the patients treated with sorafenib were hand-foot reaction and diarrhea [35]. In contrast, in the present study, mice death did not occur and no significant decrease of mice body weight was
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observed in CAs treatment group. These findings suggested that CAs had obvious antitumor activity and displayed few negative effects on host.
3.4 Effect of CAs on tumor infiltrating lymphocytes
Fig. 6. Infiltrating CD3+, CD4+, CD8+, and CD19+ lymphocytes in H22 tumor tissues were measured by flow cytometry. (A) The percentages of CD3+ and CD19+ cells were detected using CD3+ or CD19+ monoclonal antibody. (B) The percentages of CD4+ and CD8+ cells were detected using CD4+ or CD8+ monoclonal antibody.
Table 2
The percentages of tumor infiltrating CD3+ and CD8+ T lymphocytes in H22-bearing mice. Group
Dose (mg/kg)
CD3+ (%)
CD8+ (%)
Model group
-
77.24 ± 1.75
70.26 ± 4.79
22
CAs
50
83.67 ± 3.36*
76.09 ± 3.54
CAs
250
91.63 ± 3.47**
87.47 ± 2.48**
* p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
Tumor infiltrating lymphocytes (TILs) are frequently found in tumor tissues, which indicated tumor cells trigger immune response in the host. As primary host immune response against solid tumor, TILs play a major role in tumor progression and outcome [36]. Numerous researches showed that TILs have emerged as one of the most exciting tumor-related biomarkers in the breast cancer field, and tumors with high lymphocytic infiltration have better prognosis [37]. It also has been demonstrated that infiltration of CD4, CD8, and FoxP3 positive lymphocytes was associated with HPV-status and survival in oropharyngeal cancers [38]. Thus, TILs in the microenvironment could reflect tumor biology and predict outcome. In this study, the effects of CAs on TILs in H22 solid tumor were evaluated by FACS. CD19+ is B cell specific molecule that serves as a major co-stimulatory molecule for amplifying B cell receptor (BCR) responses. CD3+ is T cell specific molecule and involved in signal transduction. T lymphocytes could differentiate into two different subsets according to their specific surface molecules, including CD4+ helper T cells and CD8+ cytotoxic T cells [39, 40]. Our results (Fig. 6) showed that a large number of CD3+ T lymphocytes existed in tumor tissues, and the majority of them were CD8+ T lymphocytes. However, B lymphocytes were almost non-existent in tumor tissues. These data indicated that infiltrating CD8+ T lymphocytes were correlated with antitumor immune response in H22-bearing mice. Previous
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studies showed CD8+ TILs mediated directly tumor-associated antigen recognition and cytotoxic activity, which were able to lyse tumor cells and destroy large tumor mass in vivo. Therefore, the favorable clinical significance of CD8+ TILs has been established in several tumor types [41, 42]. In this work, the percentages of infiltrating CD3+ and CD8+ T lymphocytes were also recorded in Table 2. CAs significantly enhanced the proportions of CD3+ and CD8+ TILs compared with model group and presented a dose-dependent relationship (p < 0.01). Nevertheless, numbers, cell types and location of TILs have shown prognostic importance in cancer patients and have been associated with response to chemotherapy and prognosis [38]. Therefore, it still needs to further explore the roles of CD8+ TILs in H22 solid tumor.
3.5 CAs induced tumor cell apoptosis
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Fig. 7. CAs induced transplanted H22 cells apoptosis in vivo. (A) Tumor cell cycle was analyzed by flow cytometry. (B) Effects of CAs on the histopathological examinations of tumor tissues were observed by H&E staining (magnification, × 20). The white arrows in CAs groups represent apoptotic tumor cells nucleus bodies.
Apoptosis induction could be triggered by cell cycle arrest in tumor cells. Numerous researches showed that many antitumor drugs blocked cell cycle at a specific checkpoint and thereby induced cell apoptosis [43]. To understand whether apoptosis pathway was involved in CAs-induced H22 cells apoptosis, cell cycle was analyzed by flow cytometry. DNA contents detection (Fig. 7A) showed CAs treatment resulted in a dose-dependently accumulation of cell numbers in S phase with an increase from 27.60% to 50.84%, and a concomitant decline in the G0/G1 phase and G2/M phase. Sub-G1 DNA contents could represent percentages of apoptotic cells [3], our data showed that CAs elevated the numbers of apoptotic cell nucleus in a dose-dependent manner. These results indicated that CAsinduced suppression on H22 solid tumor was possibly mediated by blocking cell cycle in S phase.
H&E staining of tumor tissues was observed at ×20 magnification to evaluate the pathological changes of each group. The results (Fig. 7B) showed that tumor cells of model group were increased in number and volume, and had significant morphological characteristics. Consistently, the volume of tumor cellular nucleus in model group was also increased compared to CAs groups. In contrast, they were decreased in varying degrees in CAs groups. CAs leaded to chromatin condensation, nuclear shrinkage, decreased number of 25
tumor cells in a unit area, and attenuated signaling of cytoplasm staining [28]. As arrows pointed out in Fig. 7B, with the increasing concentration of CAs, tumor cellular nucleus disintegrated and formed many nuclear fragments. Thus, the result of H&E staining revealed that tumor cells were broken and the percentages of apoptotic cells were increased, which further verified that CAs could suppress growth and development of tumor cells effectively.
3.6 Effect of CAs on expression of apoptosis-related protein in tumor tissue
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Fig. 8. Effects of CAs on Bcl-xL, Mcl-1, and activated caspase 3 expression in tumor tissues were measured by western blot, and the grayscale ratio of apoptosis-related proteins relative to β-actin protein was analyzed by Quantity One software. * p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
Recently, transducer and activator of transcription 3 (STAT3) has received more and more attentions since it appears to be an important convergent point for a series of growth factors and pro-inflammatory cytokines which are involved in liver damage and contribute to liver carcinogenesis. The critical and essential roles of STAT3 in the onset and progression of liver cancer have already been demonstrated [44]. Moreover, STAT3 was found to be activated constitutively in the majority of HCC patients with poor prognosis but not in nontumor surrounding tissues or normal hepatocytes. [45, 46]. Anti-apoptotic genes such as BclxL and Mcl-1 are typical STAT3 responsive genes. Thus, the protein levels of them were measured by western blot. The result (Fig. 8) showed that the expression of Bcl-xL and Mcl-1 substantially decreased after CAs treatment compared to model group (p < 0.01). This result was in agreement with the report that kurarinol injection potentially induced transplanted H22 cells apoptosis through suppressing STAT3 responsive genes including Bcl-xL and Mcl-1 [11]. Additionally, total flavonoids from P. amplexicaule (TFPA) were also reported that it had capacity of declining the transcription and translation levels of Bcl-xL and Mcl-1 in vivo and vitro [47].
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Caspases, represented by a family of cysteine proteases, are key proteins that modulate apoptosis response. In a caspase-cascade system, caspase 8 and caspase 9 act as apoptotic initiators, whereas caspase 3 and 7 serve as apoptotic effectors. In particular, the activation of caspase 3 is one of the most common executioners in apoptotic process of various cells [48]. To further confirm that transplanted H22 cells were killed by apoptosis, the expression of activated caspase 3 was detected by western blot. The results (Fig. 8) showed that CAs dramatically enhanced the protein level of activated caspase 3 in a dose-dependent manner (p < 0.01), indicating that one of the mechanisms of CAs pro-apoptotic was achieved by increasing the expression of caspase 3. As CAs exhibited few cytotoxic effects against H22 cells in vitro (data not shown), it was supposed that the stimulation effect of CAs on caspase 3 was an indirect process in tumor-bearing mice. Previous data showed CAs increased the levels of infiltrating CD8+ T lymphocytes in tumor tissues. Thus, the activation of caspase 3 might be caused through the stimulation of immune response on host. Accordingly, the research of G. Shu et al. [28] showed that S. chinensis polysaccharides elevated cytotoxicity of natural killer and CD8+ T cells and led to the activation of caspase 3 through JAK3/STAT5 pathway in spleen tissues. However, it still needs to further explore the detailed caspasecascade system and the apoptosis pathway in CAs-induced transplanted H22 cells apoptosis.
Our current study identified that oral administration of CAs could inhibit the growth of transplanted H22 cells with few toxic impacts on host animals. Further studies showed that CAs was able to improve the levels of infiltrating CD8+ T lymphocytes, arrest tumor cell cycle in S phase, and induce tumor cells apoptosis by decreasing the expression of Bcl-xL and
28
Mcl-1and activating caspase 3 in vivo. These data provide rational explain for the traditional application of Citrus aurantifolia peels and support CAs as an adjuvant and chemopreventive compound reagent in clinic hepatocellular carcinoma treatment.
4 Conclusion
In this work, an acidic polysaccharide (CAs) was obtained from Citrus aurantifolia peels. UV and IR spectrum of CAs showed typical characteristics of acidic polysaccharide compounded with polypeptide or protein. The average molecular weight of CAs was about 7.94 × 106 Da and it was composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glu), mannose (Man) and galacturonic acid (GalA), with the molar ratio of 0.67: 7.67: 10.83: 3.83: 4.00: 1.00. 1H and 13C NMR spectrum of CAs further proved the presence of five kinds of monosaccharide and galacturonic acid. Moreover, the antitumor activity of CAs showed that it had the potential to inhibit transplanted H22 cells growth in vivo with few negative impacts on host. In addition, CAs could enhance the levels of tumor infiltrating CD8+ T lymphocytes, arrest cell cycle in S phase, and induce transplanted H22 cells apoptosis by down-regulating the levels of Bcl-xL and Mcl-1 and activating the expression of caspase 3. These data suggested that CAs had obvious antitumor activity in vivo and supported considering the application of traditional herb medicine in clinic hepatocellular carcinoma treatment.
References
[1] M.S. Grandhi, A.K. Kim, S.M. Ronnekleiv-kelly. I.R. Kamel, M.A. Ghasebeh, T.M.
29
Pawlik, Surg Oncol. 25 (2016) 74-85. [2] Y. Zhang, Q. Li, J. Wang, F. Cheng, X. Huang, Y. Cheng, K. Wang, Cancer Lett. 377 (2016) 117-125. [3] G. Shu, J. Yang, W. Zhao, C. Xu, Z. Hong, Z. Mei, X. Yang, Toxicol Appl Pharm. 281 (2014) 157-165. [4] J. Liu, S. Willför, C. Xu, Bioactive Carbohydrates and Dietary Fibre. 5 (2015) 3161. [5] A. Zong, H. Cao, F. Wang, Carbohydr Polym. 90 (2012) 1395-1410. [6] C.K. Chan, H. Supriady, B.H. Goh, H.A. Kadir, J Ethnopharmacol. 168 (2015) 291304. [7] S. Zhang, G. Kuang, G. Zhao, X. Wu, C. Zhang, R. Lei, T. Xia, J. Chen, Z. Wang, R. Ma, B. Li, L. Yang, A. Wang, Food Chem Toxicol. 62 (2013) 699-706. [8] W.D. Ma, Y.P. Zou, P. Wang, X.H. Yao, Y. Sun, M.H. Duan, Y.J. Fu, B. Yu, Food Chem Toxicol. 70 (2014) 1-8. [9] W. Song, Y. Jia, Y. Fan, M. Du, A. Liu, J Funct Foods. 5 (2013) 1270-1278.
[10] J.T. Li, J.L. Zhang, H. He, Z.L. Ma, Z.K. Nie, Z.Z. Wang, X.G. Xu, Food Chem Toxicol. 51 (2013) 297–305. [11] G. Shu, J. Yang, W. Zhao, C. Xu, Z. Hong, Z. Mei, X. Yang, Toxicol Appl Pharm. 281 (2014) 157–165.
[12] J. Kaur, G. Kaur, J Funct Foods. 13 (2015) 239-261.
30
[13] Q.H. Wang, Z.P. Shu, B.Q. Xu, N. Xing, W.J. Jiao, B.Y. Yang, H. X. Kuang, Int J Biol Macromol. 67 (2014) 112-123. [14] N.E. Rawson, C.T. Ho, S. Li, Food Science and Human Wellness. 3 (2014) 104109. [15] J.Y. Parka, M.S. Shinb, S.N. Kim, H.Y. Kim, K.H. Kim, K.S. Shin, Int J Biol Macromol. 85 (2016) 522–529. [16] E.H. Lee, H.R. Park, M.S. Shin, S.Y. Cho, H.J. Choi, K.S. Shin, Carbohydr Polym. 111 (2014) 72-79. [17] S. Moufida, B. Marzouk, Phytochemistry. 62 (2003) 1283-1289. [18] R.K. Pathan, P.R. Gali, P. Pathan, T. Gowtham, S. Pasupuleti, Asian Pac J Trop Med. S (2012) 328-331.
[19] N. Alam, P.C. Gupta, Planta Medica. 52 (1986) 308-310. [20] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal Biochem. 28 (1956) 350-356. [21] M.M. Bradford, Anal Biochem. 72 (1976) 248-254.
[22] T. Bitter, H.M. Muir, Anal Biochem. 4 (1962) 330-334. [23] Y. Wang, Y. Li, Y. Liu, X. Chen, X. Wei, Int J Biol Macromol. 77 (2015) 76-84. [24] H. Zhang, H.F.W., Y. Liu, L.J. Huang, Z.F. Wang, Y. Li, J Ethnopharmacol. 154 (2014) 818–824 [25] Y.l. Hua, W.X., M. Zhang, Y.M. Wei, P. Ji, J Ethnopharmacol. 151 (2014) 1090– 1099. 31
[26] S. Lee, J. Ra, J.Y. Song, C. Gwak, H.J. Kwon, S.V. Yim, S.P. Hong, J. Kim, K.H. Lee, J.J. Cho, Y.S. Park, C.S. Park, H.J. Ahn, J Ethnopharmacol. 133 (2011) 973– 979. [27] K.X. Cai, L.Y. Tse, C. Leung, P.K. Tam, R. Xu, M.H. Sham, Clin Cancer Res. 14 (2008) 939-949. [28] G. Shu, W. Zhao, L. Yue, H. Su, M. Xiang, J Ethnopharmacol. 168 (2015) 237247. [29] H. Liu, Y. Fan, W. Wang, N. Liu, H. Zhang, Z. Zhu, Int J Biol Macromol. 51 (2012) 417-422. [30] G.H. Mao, Y. Ren, W.W. Feng, Q. Li, H.Y. Wu, D. Jin, T. Zhao, C.Q. Xu, L.Q. Yang, X.Y. Wu, Carbohydr Polym. 134 (2015) 406–412. [31] Q. Zhu, J. Chen, Q. Li, T. Wang, H. Li, Int J Biol Macromol. 92 (2016) 156–158. [32] C. Wang, Y. Wang, J. Zhang, Z. Wang, J Taiwan Inst Chem E. 45 (2014) 1126–1132.
[33] X. Xie, G. Zou, C. Li, Carbohydr Polym. 132 (2015) 323–329. [34] G.H. Mao, Y. Ren, W.W. Feng, Q. Li, H.Y. Wu, D. Jin, T. Zhao, C.Q. Xu, L.Q. Yang, X.Y. Wu, Carbohydr Polym. 134 (2015) 406-412. [35] A.L. Cheng, Y.K. Kang, Z. Chen, C.J. Tsao, S. Qin, J.S. Kim, R. Luo, J. Feng, S. Ye, T.S. Yang, J. Xu, Y. Sun, H. Liang, J. Liu, J. Wang, W.Y. Tak, H. Pan, K. Burock, J. Zou, D. Voliotis, Z. Guan, Lancet Oncol. 10 (2009) 25-34. [36] F. Teng, X. Meng, L. Kong, D. Mu, H. Zhu, S. Liu, J. Zhang, J. Yu, Transl Res. 166 (2015) 721-32
32
[37] H.A. Jr, A. Vingiani, F. Peccatori, G. Viale, S. Loi, G. Pruneri, Breast. 24 (2015) 290-293. [38] G.T. Wolf, D.B. Chepeha, E. Bellile, A. Nguyen, D. Thomas, J. McHugh, Oral Oncol. 51 (2015) 90-95. [39] J. Jason, K.L. Inge, Hum Immunol. 6 (2000) 202–211.
[40] H. Morbach, J.N. Schickel, C. Cunningham-Rundles, M.E. Conley, I. Reisli, J.L. Franco, E. Meffre, J Allergy Clin Immunol. 137 (2016) 889-898. [41] R.F. Wang, TRENDS in Immunology. 22 (2001) 269-276. [42] Q. Zhang, Q. Jia, T. Deng, B. Song, L. Li, Biochem Biophys Res Commun. 458 (2015) 70-76. [43] C.G. Zhang, J.C. Huang, T. Liu, X.Y. Li, Original Article. 20 (2015) 1592-1600. [44] G. He, G.Y. Yu, V. Temkin, H. Ogata, C. Kuntzen, T. Sakurai, W. Sieghart, M. Peck-Radosavljevic, H.L. Leffert, M. Karin, Cancer Cell. 17 (2010) 286–297. [45] D.F. Calvisi, S. Ladu, A. Gorden, M. Farina, E.A. Conner, J.S. Lee, Gastroenterology. 130 (2006) 1117–1128. [46] L. Lin, R. Amin, G. Gallicano, E. Glasgow, W. Jogunoori, J. Jessup, M. Zasloff, J. Marshall, K. Shetty, L. Johnson, L. Mishra, A. He, Oncogene. 28 (2009) 961–972. [47] M. Xiang, H. Su, Z. Hong, T. Yang, G. Shu, Food Chem Toxicol. 80 (2015) 62– 71. [48] J.S. Lee, E.K. Hong, Cancer Lett. 297 (2010) 144-154. Figure Caption
33
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Fig. 1. The elution curve of polysaccharide fractions on Sephadex G-150. OD value at 490 nm states the content of carbohydrate by phenol-sulfuric acid method. The main fraction was named CAs.
35
36
Fig. 2. Spectra analysis of CAs. (A) UV spectrum of CAs in wavenumber region between 200 and 900 nm. (B) IR spectrum of CAs in the wavenumber region between 4000 and 400 cm-1.
37
Fig. 3. Characterization of CAs. (A) The molecular weight of CAs was determined by HPGPC analysis. (B) The monosaccharide composition of CAs was measured by IC analysis. (a) Standard samples (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, gluconic acid, galacturonic acid arranged according to the peak time); (b) CAs
38
sample (rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid arranged according to the peak time).
39
Fig. 4. NMR spectra of CAs. (A) 1H NMR spectrum; (B) 13C NMR spectrum.
40
Fig. 5. The antitumor effect of CAs on H22-bearing mice. (A) The tumor pictures of mice in model and CAs groups. (B) Effect of CAs on the tumor weight of mice. (C) Effect of CAs on the tumor volume of mice. Fig. 5(A) was taken by a digital camera (PowerShot SD 630, Canon), and the tumor size was measured using a caliper. ** p < 0.01 was very significantly different compared to model group.
Fig. 6. Infiltrating CD3+, CD4+, CD8+, and CD19+ lymphocytes in H22 tumor tissues were measured by flow cytometry. (A) The percentages of CD3+ and CD19+ cells were detected using CD3+ or CD19+ monoclonal antibody. (B) The percentages of CD4+ and CD8+ cells were detected using CD4+ or CD8+ monoclonal antibody.
41
Fig. 7. CAs induced transplanted H22 cells apoptosis in vivo. (A) Tumor cell cycle was analyzed by flow cytometry. (B) Effects of CAs on the histopathological examinations of
42
tumor tissues were observed by H&E staining (magnification, × 20). The white arrows in CAs groups represent apoptotic tumor cells nucleus bodies.
43
Fig. 8. Effects of CAs on Bcl-xL, Mcl-1, and activated caspase 3 expression in tumor tissues were measured by western blot, and the grayscale ratio of apoptosis-related proteins relative to β-actin protein was analyzed by Quantity One software. * p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
44
Table 1
The effects of CAs on body weight, number, and tumor inhibitory rate (TIR) of H22-bearing mice Group
Dose
Mice body weight (g)
Number of mice
TIR
(mg/kg)
Start
End
Start/end
(%)
Blank group
-
22.92 ± 1.01
31.91 ± 1.61**
10/ 10
Model group
-
22.22 ± 1.34
28.07 ± 1.38
10/ 9
CAs
50
22.81 ± 1.62
29.73 ± 1.37
10/ 10
29.28
CAs
250
22.00 ± 2.32
30.88 ± 1.65*
10/ 10
58.85
* p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
45
Table 2
The percentages of tumor infiltrating CD3+ and CD8+ T lymphocytes in H22-bearing mice. Group
Dose (mg/kg)
CD3+ (%)
CD8+ (%)
Model group
-
77.24 ± 1.75
70.26 ± 4.79
CAs
50
83.67 ± 3.36*
76.09 ± 3.54
CAs
250
91.63 ± 3.47**
87.47 ± 2.48**
* p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
46
S0141-8130(16)32520-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.149 BIOMAC 7315
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19-11-2016 14-3-2017 19-3-2017
Please cite this article as: Yana Zhao, Hongyan Sun, Ling Ma, Anjun Liu, Polysaccharides from the peels of Citrus aurantifolia induce apoptosis in transplanted H22 cells in mice, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.149 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Polysaccharides from the peels of Citrus aurantifolia induce apoptosis in transplanted H22 cells in mice Yana Zhao, Hongyan Sun, Ling Ma, Anjun Liu*
Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
* Corresponding author.
Tel./fax: +86 22 60912390
E-mail address: [email protected]
Highlights:
An acidic polysaccharide (CAs) with the molecular weight of 7.94 × 106 Da, which mainly composed of rhamnos, arabinose, galactose, glucose, mannose and galacturonic acid, was obtained from Citrus aurantifolia peels.
CAs had capacity of repressing transplanted H22 cells growth in vivo and exhibiting few toxic effects on host animals.
CAs was able to improve the levels of tumor infiltrating CD8+ T lymphocytes, block cell cycle in S phase, trigger apoptosis-related proteins expression and induce transplanted H22 cells apoptosis in vivo.
Abstract
In this study, an acidic polysaccharide (CAs) was extracted and purified from the peels of Citrus aurantifolia by Sephadex G-150. HPGPC showed the molecular weight of CAs was about 7.94 × 106 Da. Ion chromatography (IC) analysis showed CAs was mainly composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glu), mannose (Man) and galacturonic acid 1
(GalA), with the molar ratio of 0.67: 7.67: 10.83: 3.83: 4.00: 1.00. 1H and 13C NMR spectra of CAs also identified the presence of five kinds of monosaccharides and galacturonic acid. Moreover, the antitumor activity of CAs was evaluated in mice transplanted H22 hepatoma cells. It was shown that CAs dose-dependently suppressed tumor cells growth with few toxic effects on host. Further investigations revealed that CAs increased the levels of tumor infiltrating CD8+ T lymphocytes, blocked tumor cell cycle in S phase, down-regulated anti-apoptotic protein Bcl-xL and Mcl-1 expression, and led to the activation of caspase 3. These results suggested that CAs had capacity of inducing tumor cells apoptosis in vivo, and it supported considering CAs as an adjuvant reagent in hepatocellular carcinoma treatment.
Keywords : Citrus aurantifolia peels; Polysaccharides; Hepatocellular carcinoma.
1 Introduction
Primary liver cancer is the most common malignant tumor overall and the second most common cause of cancer mortality worldwide. Hepatocellular carcinoma (HCC) accounts for up to 90% of all primary hepatic malignancies and represents a major health problem. Surgical resection is generally regarded as the most effective strategy for treating HCC. However, more than 80% of HCC patients are not eligible to liver transplantation, surgical resection, or liver regional therapy due to advanced disease or poor liver function [1, 2]. Therefore, systemic chemotherapy is beneficial for the majority of patients. Although several compounds such as 5-fluorouracil (5-FU), sorafenib, and cisplatin have displayed a potential antitumor effect and have been already approved for HCC treatment, their prognosis is often
2
impaired by the drug resistance of HCC cells and there are a series of toxicity impacts on the normal cells and organs of patients [3]. Thus, it is urgent to search for new chemicals capable of provoking tumor cells death but exhibiting low adverse effects on the patients. Recently, polysaccharides and polysaccharide-protein complexes from traditional Chinese plants have been used as potential antitumor therapeutic agents, and their obvious antitumor activity and relatively low toxicity have been constantly confirmed [4, 5].
Apoptosis is an evolutionarily conserved program for cell self-destruction in multicellular organism indispensable for many biological processes such as development, growth, and homeostasis. It could be characterized by molecular features including chromatin condensation, DNA fragmentation, membrane blebbing, cell shrinkage and formation of apoptotic body [6, 7]. To date, the extrinsic death receptor pathway and the intrinsic mitochondrial pathway are two main apoptosis pathways, and they could be triggered by various chemical, physical and biological signals, such as chemical reagents, free radicals, radiation, and virus infection et al [8, 9]. Currently, most of therapeutic reagents achieve the purpose of antitumor through improving host immune response, suppressing tumor cell growth, arresting cell cycle at a specific phase and thereby inducing cell apoptosis in vivo or vitro. For example, corn peptides could induce Hep G2 cells apoptosis through mitochondrial apoptosis pathway in vitro and enhance host immune response in H22 tumor-bearing mice [10]. Kurarinol induced HCC cell apoptosis through repressing cellular signal transducer and activator of transcription 3 (STAT3) in vitro and vivo [11]. Therefore, the induction of
3
apoptosis has been verified as a promising strategy for the prevention and treatment of hepatocellular carcinoma.
Numbers of researches have showed that citrus peels are a promising phytochemical source with a great deal of functional components, such as phenolic, limonoids, flavonoids and polysaccharides. In some regions of the world, especially in East-Asia including China, citrus peels have been used for traditional herbal medicine as antioxidant, antibacterial, antiinflammatory and antitumor [12, 13]. Nobiletin is a fraction of Citrus flavonoid, which has been demonstrated anti-HCC activity both in vitro and vivo [14]. Polysaccharides from Korean Citrus hallabong peels inhibit angiogenesis and breast cancer cell migration in vitro [15]. It also reported that pectic polysaccharides from the peels of Korean Citrus Hallabong have obvious antitumor activity via stimulation of macrophages and NK cells [16]. Citrus aurantifolia is commonly called as key lime or bitter orange and belongs to the Rutaceae Family. C. aurantifolia peels are main by-product and are usually discarded in processing citrus. Nevertheless, the extracts from C. aurantifolia peels are an attractive source of biochemicals (e.g., flavonoids, pectin, essential oil, polysaccharides) with potential health benefits [17, 18]. However, there are few reports on the composition and biological activity of C. aurantifolia peels polysaccharides. In this work, an acidic polysaccharide was isolated and purified from the peels of C. aurantifolia. Moreover, antitumor activity and possible mechanism of the polysaccharide against transplanted H22 cells were also evaluated in vivo.
2 Materials and methods
4
2.1 Materials and reagents
Citrus aurantifolia was obtained from Haikou City, China. D-fucose, D-rhamnose, Larabinose, D-xylose, D-mannose, D-glucose, D-galactose, D-fructose, D-gluconic acid, Dgalacturonic acid, T-series dextran standards, propidium iodine (PI) and RIPA lysis buffer were purchased from Solarbio Co. (Beijing, China). Sephadex G-150 and bovine albumin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bradford Protein Assay Kit was purchased from Nanjing Jiancheng Biotech. Co. (Jiangsu, China). Primary antibodies were purchased from Tianjin Sungene Biotech Co. (Tianjin, China). Secondary antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA). The other chemical regents were of analytical grade.
2.2 Extraction, separation and purification of polysaccharides
C. aurantifolia peels were separated and dried in an oven at 40 ℃ until constant weight, then crushed into powder (particle size: 0.6 mm). The powder was refluxed with petroleum ether for 2 h to remove some colored materials, grease and other low molecular compounds. Then the residue was dried in air and extracted with 0.3 M NaOH (ratio of solid to liquid = 1:10) for three times (2 h each time) at room temperature. The supernatant was collected and the value of pH was adjusted to 3.0 using 0.1 M HCl, following precipitated by 80% (v/v) anhydrous ethanol and incubated at 4 ℃ overnight to obtain crude polysaccharides by centrifugation (3500 × g, 20 min).
5
The crude polysaccharides were deproteinated using n-butanol and chloroform (1:4, v/v) according to the method of Sevage [19]. Then the mixture was concentrated, dialyzed (Mw 3500) with tap water for 48 h and distilled water for 48 h and lyophilized. The gained polysaccharides were further purified by Sephadex G-150 chromatography (2.6 cm × 35 cm). The column was eluted with distilled water at 0.8 ml/min and the polysaccharide fractions were collected by detecting carbohydrate with phenol-sulfuric acid method [20]. An acidic polysaccharide fraction was obtained and designated as CAs, which was used for next investigation.
2.3 Physicochemical properties of CAs
2.3.1 Ultraviolet (UV) and infrared (IR) analysis
CAs was dissolved with distilled water and diluted to proper concentration, then scanned from 200 to 900 nm with a Genesys 10s UV-VIS spectrophotometer (Thermo Scientific, America). IR spectrum of CAs was detected with a Fourier transformed infrared spectrophotometer (Bruker Vector-220, German) in the wave range of 4000 to 400 cm-1 using the KBr-disk method.
2.3.2 Chemical components analysis
Total carbohydrate content in CAs was tested by phenol-sulfuric acid method with galactose as standard [20]. Protein content in CAs was measured according to coomassie brilliant blue method using BSA as standard [21]. Uronic acid content in CAs was quantified by sulfuric acid-carbazole method using galacturonic acid as reference [22].
6
2.3.3 Homogeneity and molecular weight analysis
The homogeneity and molecular weight of CAs was determined by high-performance gel-permeation chromatography (HPGPC) (Agilent-1200) equipped with a Tsk-gel G4000PWxl column (7.8 mm × 300 mm, column temperature 30 ℃) and Refractive Index Detector (RID, detecting temperature 35 ℃). 20 ul of polysaccharide solution was eluted at a flow rate of 0.8 ml/min with distilled water. The standard curve was established with T-series dextran (T-10, 40, 70, 110, 500 and 2000) as standard, and the molecular weight of CAs was calculated according to the calibration curve.
2.3.4 Monosaccharide composition analysis
The monosaccharide composition of CAs was detected by ion chromatography (IC), performed on a Dionex ISC5000 chromatographic system (CA, USA) with an efficient anion exchange column of Dionex Carbopac PA20 column (150 mm × 3 mm) and a Dionex pulsed amperometric detector equipped with an Au electrode. The polysaccharide was hydrolyzed as described [23], with some modifications. Chiefly, polysaccharide (5 mg) was dissolved in 2 M trifluoroacetic acid (TFA) and hydrolyzed at 120 ℃ for 6 h. Then TFA was absolutely removed by adding methanol and N2 was used as the carrier gas. The dried hydrolysate was dissolved with deionized water. NaOH solution (10 mM) and sodium acetate solution (200 mM) were used as mobile phase with gradient elution at a flow rate of 0.5 ml/min. Fucose, rhamnose, arabinose, xylose, mannose, glucose, galactose, fructose, glucuronic acid and galacturonic acid were used as references.
7
2.3.5 Nuclear magnetic resonance (NMR) analysis
The polysaccharide was exchanged three times with deuterium in D2O by lyophilizing. 1
H and 13C NMR spectra were recorded on a NMR spectrometer (Brucker DRX-500) at 600
MHz.
2.4 Animals and animal experiments
ICR mice (female, 8 weeks old, weighting 20 ± 2 g) were purchased from the Department of Experimental Animal, Academy of Military Medical Science, Beijing, China. All the mice were under barrier conditions in the Center of Experimental Animals at Tianjin University of Science and Technology. The mice were allowed free access to food and water and were kept constant room temperature of 24 ± 1 ℃, 50 ± 10% relative humidity with a 12/12 h light/dark cycle. All animal handing procedures were performed strictly followed the Principles of Laboratory Animal Care (NIH Publication No.86-23, revised 1985).
In an array of previous studies, the experimental doses of plant polysaccharides on mice were around 100 mg/kg [24, 25]. In addition, for some functional components in citrus peels, we observed the Citrus peels flavonoids on H22-bearing mice were 125 - 500 mg/kg [14], and Citrus unshiu peel extracts on mice renal cell carcinoma model were 30 mg/kg [26]. Therefore, we selected two doses of CAs (50 and 250 mg/kg) as the low and the high dose in this study. Next, the mice were randomly divided into groups with ten mice in each group and treated as described in the text. Blank group and model group were both applied physiological saline solution orally throughout the experimental period. CAs groups were given CAs
8
solution with 50 or 250 mg/kg body weight every day. Two weeks later, all the mice in model group and CAs groups were subcutaneously injected mouse H22 ascitic hepatoma cells (obtained from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) in the right thigh of the lower limb with 0.2 ml (5 × 106 cells/ml). Then all the mice received the treatment for three weeks successively. At the end of the experiment, each group mice were weighed. The tumor volume was measured with a caliper and calculated according to following formula: Tumor volume (cm3) = LW2/2，wherein L and W stand for tumor length and width, respectively [27]. Then all the mice were sacrificed by cervical dislocation. The tumor tissues were dissected out and immediately weighed. The tumor inhibitory rate (TIR) was calculated by the formula: Tumor inhibitory rate (%) = (W1 – W2)/W1 × 100%. Where, W1 and W2 stand for the average tumor weight of model group and CAs groups mice, respectively [28].
2.5 Tumor infiltrating lymphocytes (TILs) analysis
Tumor tissues were collected under aseptic conditions from sacrificed mice and smashed with scissors, then gently passed through sterilized steel mesh to obtain homogeneous tumor cells suspension. The cells were adjusted to 1 × 106 cells/ml and incubated with either CD3FITC, CD4-PE, CD8-FITC, or CD19-PE monoclonal antibody in dark for 30 min at 4 ℃, then washed with PBS to remove unbounded antibody. The percentages of TILs were measured by flow cytometry (BD FACSCalibur, USA), and analyzed by CellQuest Pro. software.
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2.6 Cell cycle analysis
Tumor cells suspension was obtained as described previously. The cells were washed with PBS and adjusted to 1 × 106 cells/ml, then fixed with cold 70% ethanol (stored at -20 ℃) overnight. After washing, intracellular RNA was removed by incubating samples with RNase A (0.1 mg/ml) at 37 ℃ for 30 min. Then they were stained with PI (50 μg/ml) in dark for 30 min at 37 ℃. Cell cycle was detected by flow cytometry (BD FACSCalibur, USA), and analyzed by ModFit LT software.
2.7 Histology analysis
Tumor tissues were fixed with 10% (v/v) buffered formalin, dehydrated through a graded series of ethanol, cleared in xylene and embedded in paraffin. Slices (5-6 μm thick) of tumor tissues were prepared from paraffin blocks and stained with hematoxylin-eosin (H&E). Histopathological changes were examined by microscopy.
2.8 Western Blot analysis
Tumor tissues were lysed in RIPA lysis buffer according to manufacture’s instruction. The supernatant was collected and centrifuged at 10000 g for 5 min. Concentrations of proteins were quantified using Bradford Protein Assay Kit. The supernatant proteins were mixed with 1 × reducing electrophoresis loading buffer and boiled for 5 min. Protein samples (50-70 μg) were separated by 12% SDS-PAGE and transferred to PVDF membrane at 15 V by semi-dry blotting. Membranes were blocked with 5% bovine albumin in TBST for 2 h at room temperature and incubated with primary antibody overnight at 4 ℃. Primary antibodies
10
were β-actin antibody (1:10000), Bcl-xL antibody (1:1000), Mcl-1 antibody (1:1000), and caspase 3 antibody (1:1000) were diluted in blocking solution. Membranes were washed with TBST three times (5 min/wash), and further incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-mouse IgG 1:10000 or goat antirabbit IgG 1:5000) for 1.5 h at room temperature. Finally, protein bands were examined using chemiluminescence (ECL) reagents and exposed to X-ray photographic films in a darkroom. The bands were visualized and analyzed with Quantity One software.
2.9 Statistical analysis
All experiments were performed at least three times. Experimental data were presented as mean ± standard deviation. Significant differences were established through analysis of variance (ANOVA) and mean comparisons were achieved by Duncan’s multiple range test. Data analysis was evaluated using SPSS software (SPSS Inc., Chicago, IL, USA). *p < 0.05 was considered as significant, and **p < 0.01 was considered as very significant.
3 Results and discussion
3.1 Isolation and purification of polysaccharide
11
Fig. 1. The elution curve of polysaccharide fractions on Sephadex G-150. OD value at 490 nm states the content of carbohydrate by phenol-sulfuric acid method. The main fraction was named CAs.
The crude polysaccharide was obtained from the dried C. aurantifolia peels powder, and the yield of dry material was about 2.89%. As shown in Fig. 1, the crude polysaccharide was further separated by Sephadex G-150 and two fractions were gained. The second fraction was excluded in the following structure characterization due to the presence of some colored materials and low carbohydrate content based on the phenol-sulfuric acid assay. Thus, main product was collected and named CAs.
3.2 Chemical characterization of CAs
12
Fig. 2. Spectra analysis of CAs. (A) UV spectrum of CAs in wavenumber region between 200 and 900 nm. (B) IR spectrum of CAs in the wavenumber region between 4000 and 400 cm-1.
13
14
Fig. 3. Characterization of CAs. (A) The molecular weight of CAs was determined by HPGPC analysis. (B) The monosaccharide composition of CAs was measured by IC analysis. (a) Standard samples (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, gluconic acid, galacturonic acid arranged according to the peak time); (b) CAs sample (rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid arranged according to the peak time).
15
Fig. 4. NMR spectra of CAs. (A) 1H NMR spectrum; (B) 13C NMR spectrum.
The UV spectrum analysis (Fig. 2A) showed that CAs had a weak absorption at 280 nm, indicating the presence of protein in polysaccharide. The IR spectrum of CAs was showed in Fig. 2B, which revealed classical polysaccharide absorbance peaks. The broadly intense peak
16
at 3416 cm-1 was the stretching vibration of hydroxyl groups, and weak band at 2925 cm-1 belonged to the saturated C-H stretching vibration. The band at 1628 cm-1 due to N-H (CONH-) variable angle vibration, suggesting the presence of binding protein in polysaccharide [8]. The band at 1416 cm-1 was ascribed to -COOH stretching vibration. The intense peak at 1050 cm-1 was assigned the C-O-C glycosidic linkage. The peak at 896 cm-1 was attributed to the presence of α-D-pyranoid glucose, and the peak at 777 cm-1 may suggest the presence of α-isomers of pyranose [29].
The carbohydrate content of CAs was about 80.33% based on the phenol-sulfuric acid method. The protein content was about 4.36% by the Coomassie brilliant blue method. The uronic acid content of CAs was about 10.56% based on the sulfuric acid-carbazole method. The results showed that a small quantity of polypeptides or protein combined with polysaccharide.
The HPGPC analysis (Fig. 3A) showed that CAs only had one main peak with the retention time of 7.931, indicating that it was a homogeneous polysaccharide and the average molecular weight was 7.94 × 106 Da calculated in accordance with the retention time of standard dextrans. The standard curve of molecular weight was Y= -0.4367X + 10.429, R2 = 0.9902 (Y = log Mw, X = Rt). Analysis of monosaccharide composition of CAs using IC (Fig. 3B) showed that it was composed of rhamnose, arabinose, galactose, glucose, mannose and galacturonic acid, with the molar ratio of 0.67: 7.67: 10.83: 3.83: 4.00: 1.00.
17
The structural features of CAs were further verified by NMR spectra analysis (Fig. 4). The chemical shifts at 5.020 - 5.375 ppm in the 1H NMR spectrum could be assigned to the typical signals of anomeric protons. The four anomeric protons signals at 5.375 (5.332), 5.186, 5.114 (5.084), 5.020 indicated CAs was mainly composed of glucose, galactose, mannose and arabinose. The signal at 1.177 ppm proved the presence of methyl protons in rhamnose. In 13C NMR spectrum, the signal at 177.53 ppm showed the presence of carboxyl carbon, which supported the conclusion that CAs was consisted of galacturonic acid. The four major signals indicated that the polysaccharide was composed of four kinds of monosaccharides. Additionally, the signal at 16.71 ppm revealed the presence of rhamnose in CAs.
3.3 Effect of CAs on transplanted H22 cell growth
Table 1
The effects of CAs on body weight, number, and tumor inhibitory rate (TIR) of H22-bearing mice Group
Dose
Mice body weight (g)
Number of mice
TIR
(mg/kg)
Start
End
Start/end
(%)
Blank group
-
22.92 ± 1.01
31.91 ± 1.61**
10/ 10
Model group
-
22.22 ± 1.34
28.07 ± 1.38
10/ 9
CAs
50
22.81 ± 1.62
29.73 ± 1.37
10/ 10
29.28
CAs
250
22.00 ± 2.32
30.88 ± 1.65*
10/ 10
58.85
18
* p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
19
Fig. 5. The antitumor effect of CAs on H22-bearing mice. (A) The tumor pictures of mice in model and CAs groups. (B) Effect of CAs on the tumor weight of mice. (C) Effect of CAs on the tumor volume of mice. Fig. 5(A) was taken by a digital camera (PowerShot SD 630, Canon), and the tumor size was measured using a caliper. ** p < 0.01 was very significantly different compared to model group.
As shown in Table 1, in the initial stage of experiment, there were no significant differences of body weight among all groups. However, at the end of the experiment period, the body weight in model group obviously declined compared to blank group (p < 0.01). This might be caused by the rapidly growing tumor tissues. Meanwhile, we observed that mice in model group had thin body, slow autonomic movement and sparse hair. In contrast, the average body weight in CAs groups were no significant differences compared to blank group, and it had a significant increase compared to model group (p < 0.05), indicating that CAs treatment exhibited few negative impacts on body weight of tumor-bearing mice. Additionally, mice in CAs groups kept a normal body weight, good appetite and shiny fur. To further assess inhibitory activity of CAs, the average tumor weight and tumor volume were noted. As shown in Fig. 5, the average tumor weight and tumor volume in model group were significantly higher than CAs groups (p < 0.01). It was also shown that antitumor effect of CAs at 250 mg/kg was superior to the dose of 50 mg/kg, suggesting that the beneficial effect was performed in a dose-dependent manner. Moreover, the tumor inhibition rate (TIR) was also calculated in this work. As the results showed in Table 1, the TIR attained 29.28% and 58.85% in two doses of CAs, respectively, which revealed CAs had capacity of repressing
20
H22 solid tumor growth. Similarly, according to the research of G.H. Mao et al. [30], a watersoluble polysaccharide from Grifola frondosa significantly inhibited the growth of Heps cells in vivo and tumor inhibition rate attained 56.16%. Q. Zhu et al. [31] found that Laminaria japonica polysaccharide improved immunolomodulatory activity and decreased tumor weight in H22-bearing mice. Simultaneously, tumor inhibition rate increased in a dose-dependent manner and it reached up to 59.67%. In addition, other polysaccharides, such as from Gentiana scabra Bunge [32], and Chaenomeles speciose [33], also have been investigated S180-repressing activity in vivo, and the highest tumor inhibition rate could attain 65.76% and 44.9%, respectively. Tumor inhibition rates of these polysaccharides are similar with the antitumor activity of CAs, supporting the application of Citrus aurantifolia peels in hepatocellular carcinoma treatment.
5-FU, cisplatin and sorafenib are conventional chemotherapeutic drugs widely applied in clinical HCC treatment, which present obvious tumor-suppressing activity. The research showed that 5-FU had potential inhibitory impacts on the growth of Heps tumors, and the inhibitory rate reached 68.49% compared to negative group. However, a significant decrease in spleen and thymus relative weight was also observed in 5-FU group [34]. Consistently, it also has been reported that cisplatin showed stronger HCC-suppressing activity with a tumor inhibition rate of 79.4%. However, at the end of 10 days treatment, 2 out of 10 mice died in cisplatin group [28]. In addition, the most frequently reported the adverse events in the patients treated with sorafenib were hand-foot reaction and diarrhea [35]. In contrast, in the present study, mice death did not occur and no significant decrease of mice body weight was
21
observed in CAs treatment group. These findings suggested that CAs had obvious antitumor activity and displayed few negative effects on host.
3.4 Effect of CAs on tumor infiltrating lymphocytes
Fig. 6. Infiltrating CD3+, CD4+, CD8+, and CD19+ lymphocytes in H22 tumor tissues were measured by flow cytometry. (A) The percentages of CD3+ and CD19+ cells were detected using CD3+ or CD19+ monoclonal antibody. (B) The percentages of CD4+ and CD8+ cells were detected using CD4+ or CD8+ monoclonal antibody.
Table 2
The percentages of tumor infiltrating CD3+ and CD8+ T lymphocytes in H22-bearing mice. Group
Dose (mg/kg)
CD3+ (%)
CD8+ (%)
Model group
-
77.24 ± 1.75
70.26 ± 4.79
22
CAs
50
83.67 ± 3.36*
76.09 ± 3.54
CAs
250
91.63 ± 3.47**
87.47 ± 2.48**
* p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
Tumor infiltrating lymphocytes (TILs) are frequently found in tumor tissues, which indicated tumor cells trigger immune response in the host. As primary host immune response against solid tumor, TILs play a major role in tumor progression and outcome [36]. Numerous researches showed that TILs have emerged as one of the most exciting tumor-related biomarkers in the breast cancer field, and tumors with high lymphocytic infiltration have better prognosis [37]. It also has been demonstrated that infiltration of CD4, CD8, and FoxP3 positive lymphocytes was associated with HPV-status and survival in oropharyngeal cancers [38]. Thus, TILs in the microenvironment could reflect tumor biology and predict outcome. In this study, the effects of CAs on TILs in H22 solid tumor were evaluated by FACS. CD19+ is B cell specific molecule that serves as a major co-stimulatory molecule for amplifying B cell receptor (BCR) responses. CD3+ is T cell specific molecule and involved in signal transduction. T lymphocytes could differentiate into two different subsets according to their specific surface molecules, including CD4+ helper T cells and CD8+ cytotoxic T cells [39, 40]. Our results (Fig. 6) showed that a large number of CD3+ T lymphocytes existed in tumor tissues, and the majority of them were CD8+ T lymphocytes. However, B lymphocytes were almost non-existent in tumor tissues. These data indicated that infiltrating CD8+ T lymphocytes were correlated with antitumor immune response in H22-bearing mice. Previous
23
studies showed CD8+ TILs mediated directly tumor-associated antigen recognition and cytotoxic activity, which were able to lyse tumor cells and destroy large tumor mass in vivo. Therefore, the favorable clinical significance of CD8+ TILs has been established in several tumor types [41, 42]. In this work, the percentages of infiltrating CD3+ and CD8+ T lymphocytes were also recorded in Table 2. CAs significantly enhanced the proportions of CD3+ and CD8+ TILs compared with model group and presented a dose-dependent relationship (p < 0.01). Nevertheless, numbers, cell types and location of TILs have shown prognostic importance in cancer patients and have been associated with response to chemotherapy and prognosis [38]. Therefore, it still needs to further explore the roles of CD8+ TILs in H22 solid tumor.
3.5 CAs induced tumor cell apoptosis
24
Fig. 7. CAs induced transplanted H22 cells apoptosis in vivo. (A) Tumor cell cycle was analyzed by flow cytometry. (B) Effects of CAs on the histopathological examinations of tumor tissues were observed by H&E staining (magnification, × 20). The white arrows in CAs groups represent apoptotic tumor cells nucleus bodies.
Apoptosis induction could be triggered by cell cycle arrest in tumor cells. Numerous researches showed that many antitumor drugs blocked cell cycle at a specific checkpoint and thereby induced cell apoptosis [43]. To understand whether apoptosis pathway was involved in CAs-induced H22 cells apoptosis, cell cycle was analyzed by flow cytometry. DNA contents detection (Fig. 7A) showed CAs treatment resulted in a dose-dependently accumulation of cell numbers in S phase with an increase from 27.60% to 50.84%, and a concomitant decline in the G0/G1 phase and G2/M phase. Sub-G1 DNA contents could represent percentages of apoptotic cells [3], our data showed that CAs elevated the numbers of apoptotic cell nucleus in a dose-dependent manner. These results indicated that CAsinduced suppression on H22 solid tumor was possibly mediated by blocking cell cycle in S phase.
H&E staining of tumor tissues was observed at ×20 magnification to evaluate the pathological changes of each group. The results (Fig. 7B) showed that tumor cells of model group were increased in number and volume, and had significant morphological characteristics. Consistently, the volume of tumor cellular nucleus in model group was also increased compared to CAs groups. In contrast, they were decreased in varying degrees in CAs groups. CAs leaded to chromatin condensation, nuclear shrinkage, decreased number of 25
tumor cells in a unit area, and attenuated signaling of cytoplasm staining [28]. As arrows pointed out in Fig. 7B, with the increasing concentration of CAs, tumor cellular nucleus disintegrated and formed many nuclear fragments. Thus, the result of H&E staining revealed that tumor cells were broken and the percentages of apoptotic cells were increased, which further verified that CAs could suppress growth and development of tumor cells effectively.
3.6 Effect of CAs on expression of apoptosis-related protein in tumor tissue
26
Fig. 8. Effects of CAs on Bcl-xL, Mcl-1, and activated caspase 3 expression in tumor tissues were measured by western blot, and the grayscale ratio of apoptosis-related proteins relative to β-actin protein was analyzed by Quantity One software. * p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
Recently, transducer and activator of transcription 3 (STAT3) has received more and more attentions since it appears to be an important convergent point for a series of growth factors and pro-inflammatory cytokines which are involved in liver damage and contribute to liver carcinogenesis. The critical and essential roles of STAT3 in the onset and progression of liver cancer have already been demonstrated [44]. Moreover, STAT3 was found to be activated constitutively in the majority of HCC patients with poor prognosis but not in nontumor surrounding tissues or normal hepatocytes. [45, 46]. Anti-apoptotic genes such as BclxL and Mcl-1 are typical STAT3 responsive genes. Thus, the protein levels of them were measured by western blot. The result (Fig. 8) showed that the expression of Bcl-xL and Mcl-1 substantially decreased after CAs treatment compared to model group (p < 0.01). This result was in agreement with the report that kurarinol injection potentially induced transplanted H22 cells apoptosis through suppressing STAT3 responsive genes including Bcl-xL and Mcl-1 [11]. Additionally, total flavonoids from P. amplexicaule (TFPA) were also reported that it had capacity of declining the transcription and translation levels of Bcl-xL and Mcl-1 in vivo and vitro [47].
27
Caspases, represented by a family of cysteine proteases, are key proteins that modulate apoptosis response. In a caspase-cascade system, caspase 8 and caspase 9 act as apoptotic initiators, whereas caspase 3 and 7 serve as apoptotic effectors. In particular, the activation of caspase 3 is one of the most common executioners in apoptotic process of various cells [48]. To further confirm that transplanted H22 cells were killed by apoptosis, the expression of activated caspase 3 was detected by western blot. The results (Fig. 8) showed that CAs dramatically enhanced the protein level of activated caspase 3 in a dose-dependent manner (p < 0.01), indicating that one of the mechanisms of CAs pro-apoptotic was achieved by increasing the expression of caspase 3. As CAs exhibited few cytotoxic effects against H22 cells in vitro (data not shown), it was supposed that the stimulation effect of CAs on caspase 3 was an indirect process in tumor-bearing mice. Previous data showed CAs increased the levels of infiltrating CD8+ T lymphocytes in tumor tissues. Thus, the activation of caspase 3 might be caused through the stimulation of immune response on host. Accordingly, the research of G. Shu et al. [28] showed that S. chinensis polysaccharides elevated cytotoxicity of natural killer and CD8+ T cells and led to the activation of caspase 3 through JAK3/STAT5 pathway in spleen tissues. However, it still needs to further explore the detailed caspasecascade system and the apoptosis pathway in CAs-induced transplanted H22 cells apoptosis.
Our current study identified that oral administration of CAs could inhibit the growth of transplanted H22 cells with few toxic impacts on host animals. Further studies showed that CAs was able to improve the levels of infiltrating CD8+ T lymphocytes, arrest tumor cell cycle in S phase, and induce tumor cells apoptosis by decreasing the expression of Bcl-xL and
28
Mcl-1and activating caspase 3 in vivo. These data provide rational explain for the traditional application of Citrus aurantifolia peels and support CAs as an adjuvant and chemopreventive compound reagent in clinic hepatocellular carcinoma treatment.
4 Conclusion
In this work, an acidic polysaccharide (CAs) was obtained from Citrus aurantifolia peels. UV and IR spectrum of CAs showed typical characteristics of acidic polysaccharide compounded with polypeptide or protein. The average molecular weight of CAs was about 7.94 × 106 Da and it was composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glu), mannose (Man) and galacturonic acid (GalA), with the molar ratio of 0.67: 7.67: 10.83: 3.83: 4.00: 1.00. 1H and 13C NMR spectrum of CAs further proved the presence of five kinds of monosaccharide and galacturonic acid. Moreover, the antitumor activity of CAs showed that it had the potential to inhibit transplanted H22 cells growth in vivo with few negative impacts on host. In addition, CAs could enhance the levels of tumor infiltrating CD8+ T lymphocytes, arrest cell cycle in S phase, and induce transplanted H22 cells apoptosis by down-regulating the levels of Bcl-xL and Mcl-1 and activating the expression of caspase 3. These data suggested that CAs had obvious antitumor activity in vivo and supported considering the application of traditional herb medicine in clinic hepatocellular carcinoma treatment.
References
[1] M.S. Grandhi, A.K. Kim, S.M. Ronnekleiv-kelly. I.R. Kamel, M.A. Ghasebeh, T.M.
29
Pawlik, Surg Oncol. 25 (2016) 74-85. [2] Y. Zhang, Q. Li, J. Wang, F. Cheng, X. Huang, Y. Cheng, K. Wang, Cancer Lett. 377 (2016) 117-125. [3] G. Shu, J. Yang, W. Zhao, C. Xu, Z. Hong, Z. Mei, X. Yang, Toxicol Appl Pharm. 281 (2014) 157-165. [4] J. Liu, S. Willför, C. Xu, Bioactive Carbohydrates and Dietary Fibre. 5 (2015) 3161. [5] A. Zong, H. Cao, F. Wang, Carbohydr Polym. 90 (2012) 1395-1410. [6] C.K. Chan, H. Supriady, B.H. Goh, H.A. Kadir, J Ethnopharmacol. 168 (2015) 291304. [7] S. Zhang, G. Kuang, G. Zhao, X. Wu, C. Zhang, R. Lei, T. Xia, J. Chen, Z. Wang, R. Ma, B. Li, L. Yang, A. Wang, Food Chem Toxicol. 62 (2013) 699-706. [8] W.D. Ma, Y.P. Zou, P. Wang, X.H. Yao, Y. Sun, M.H. Duan, Y.J. Fu, B. Yu, Food Chem Toxicol. 70 (2014) 1-8. [9] W. Song, Y. Jia, Y. Fan, M. Du, A. Liu, J Funct Foods. 5 (2013) 1270-1278.
[10] J.T. Li, J.L. Zhang, H. He, Z.L. Ma, Z.K. Nie, Z.Z. Wang, X.G. Xu, Food Chem Toxicol. 51 (2013) 297–305. [11] G. Shu, J. Yang, W. Zhao, C. Xu, Z. Hong, Z. Mei, X. Yang, Toxicol Appl Pharm. 281 (2014) 157–165.
[12] J. Kaur, G. Kaur, J Funct Foods. 13 (2015) 239-261.
30
[13] Q.H. Wang, Z.P. Shu, B.Q. Xu, N. Xing, W.J. Jiao, B.Y. Yang, H. X. Kuang, Int J Biol Macromol. 67 (2014) 112-123. [14] N.E. Rawson, C.T. Ho, S. Li, Food Science and Human Wellness. 3 (2014) 104109. [15] J.Y. Parka, M.S. Shinb, S.N. Kim, H.Y. Kim, K.H. Kim, K.S. Shin, Int J Biol Macromol. 85 (2016) 522–529. [16] E.H. Lee, H.R. Park, M.S. Shin, S.Y. Cho, H.J. Choi, K.S. Shin, Carbohydr Polym. 111 (2014) 72-79. [17] S. Moufida, B. Marzouk, Phytochemistry. 62 (2003) 1283-1289. [18] R.K. Pathan, P.R. Gali, P. Pathan, T. Gowtham, S. Pasupuleti, Asian Pac J Trop Med. S (2012) 328-331.
[19] N. Alam, P.C. Gupta, Planta Medica. 52 (1986) 308-310. [20] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal Biochem. 28 (1956) 350-356. [21] M.M. Bradford, Anal Biochem. 72 (1976) 248-254.
[22] T. Bitter, H.M. Muir, Anal Biochem. 4 (1962) 330-334. [23] Y. Wang, Y. Li, Y. Liu, X. Chen, X. Wei, Int J Biol Macromol. 77 (2015) 76-84. [24] H. Zhang, H.F.W., Y. Liu, L.J. Huang, Z.F. Wang, Y. Li, J Ethnopharmacol. 154 (2014) 818–824 [25] Y.l. Hua, W.X., M. Zhang, Y.M. Wei, P. Ji, J Ethnopharmacol. 151 (2014) 1090– 1099. 31
[26] S. Lee, J. Ra, J.Y. Song, C. Gwak, H.J. Kwon, S.V. Yim, S.P. Hong, J. Kim, K.H. Lee, J.J. Cho, Y.S. Park, C.S. Park, H.J. Ahn, J Ethnopharmacol. 133 (2011) 973– 979. [27] K.X. Cai, L.Y. Tse, C. Leung, P.K. Tam, R. Xu, M.H. Sham, Clin Cancer Res. 14 (2008) 939-949. [28] G. Shu, W. Zhao, L. Yue, H. Su, M. Xiang, J Ethnopharmacol. 168 (2015) 237247. [29] H. Liu, Y. Fan, W. Wang, N. Liu, H. Zhang, Z. Zhu, Int J Biol Macromol. 51 (2012) 417-422. [30] G.H. Mao, Y. Ren, W.W. Feng, Q. Li, H.Y. Wu, D. Jin, T. Zhao, C.Q. Xu, L.Q. Yang, X.Y. Wu, Carbohydr Polym. 134 (2015) 406–412. [31] Q. Zhu, J. Chen, Q. Li, T. Wang, H. Li, Int J Biol Macromol. 92 (2016) 156–158. [32] C. Wang, Y. Wang, J. Zhang, Z. Wang, J Taiwan Inst Chem E. 45 (2014) 1126–1132.
[33] X. Xie, G. Zou, C. Li, Carbohydr Polym. 132 (2015) 323–329. [34] G.H. Mao, Y. Ren, W.W. Feng, Q. Li, H.Y. Wu, D. Jin, T. Zhao, C.Q. Xu, L.Q. Yang, X.Y. Wu, Carbohydr Polym. 134 (2015) 406-412. [35] A.L. Cheng, Y.K. Kang, Z. Chen, C.J. Tsao, S. Qin, J.S. Kim, R. Luo, J. Feng, S. Ye, T.S. Yang, J. Xu, Y. Sun, H. Liang, J. Liu, J. Wang, W.Y. Tak, H. Pan, K. Burock, J. Zou, D. Voliotis, Z. Guan, Lancet Oncol. 10 (2009) 25-34. [36] F. Teng, X. Meng, L. Kong, D. Mu, H. Zhu, S. Liu, J. Zhang, J. Yu, Transl Res. 166 (2015) 721-32
32
[37] H.A. Jr, A. Vingiani, F. Peccatori, G. Viale, S. Loi, G. Pruneri, Breast. 24 (2015) 290-293. [38] G.T. Wolf, D.B. Chepeha, E. Bellile, A. Nguyen, D. Thomas, J. McHugh, Oral Oncol. 51 (2015) 90-95. [39] J. Jason, K.L. Inge, Hum Immunol. 6 (2000) 202–211.
[40] H. Morbach, J.N. Schickel, C. Cunningham-Rundles, M.E. Conley, I. Reisli, J.L. Franco, E. Meffre, J Allergy Clin Immunol. 137 (2016) 889-898. [41] R.F. Wang, TRENDS in Immunology. 22 (2001) 269-276. [42] Q. Zhang, Q. Jia, T. Deng, B. Song, L. Li, Biochem Biophys Res Commun. 458 (2015) 70-76. [43] C.G. Zhang, J.C. Huang, T. Liu, X.Y. Li, Original Article. 20 (2015) 1592-1600. [44] G. He, G.Y. Yu, V. Temkin, H. Ogata, C. Kuntzen, T. Sakurai, W. Sieghart, M. Peck-Radosavljevic, H.L. Leffert, M. Karin, Cancer Cell. 17 (2010) 286–297. [45] D.F. Calvisi, S. Ladu, A. Gorden, M. Farina, E.A. Conner, J.S. Lee, Gastroenterology. 130 (2006) 1117–1128. [46] L. Lin, R. Amin, G. Gallicano, E. Glasgow, W. Jogunoori, J. Jessup, M. Zasloff, J. Marshall, K. Shetty, L. Johnson, L. Mishra, A. He, Oncogene. 28 (2009) 961–972. [47] M. Xiang, H. Su, Z. Hong, T. Yang, G. Shu, Food Chem Toxicol. 80 (2015) 62– 71. [48] J.S. Lee, E.K. Hong, Cancer Lett. 297 (2010) 144-154. Figure Caption
33
34
Fig. 1. The elution curve of polysaccharide fractions on Sephadex G-150. OD value at 490 nm states the content of carbohydrate by phenol-sulfuric acid method. The main fraction was named CAs.
35
36
Fig. 2. Spectra analysis of CAs. (A) UV spectrum of CAs in wavenumber region between 200 and 900 nm. (B) IR spectrum of CAs in the wavenumber region between 4000 and 400 cm-1.
37
Fig. 3. Characterization of CAs. (A) The molecular weight of CAs was determined by HPGPC analysis. (B) The monosaccharide composition of CAs was measured by IC analysis. (a) Standard samples (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, gluconic acid, galacturonic acid arranged according to the peak time); (b) CAs
38
sample (rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid arranged according to the peak time).
39
Fig. 4. NMR spectra of CAs. (A) 1H NMR spectrum; (B) 13C NMR spectrum.
40
Fig. 5. The antitumor effect of CAs on H22-bearing mice. (A) The tumor pictures of mice in model and CAs groups. (B) Effect of CAs on the tumor weight of mice. (C) Effect of CAs on the tumor volume of mice. Fig. 5(A) was taken by a digital camera (PowerShot SD 630, Canon), and the tumor size was measured using a caliper. ** p < 0.01 was very significantly different compared to model group.
Fig. 6. Infiltrating CD3+, CD4+, CD8+, and CD19+ lymphocytes in H22 tumor tissues were measured by flow cytometry. (A) The percentages of CD3+ and CD19+ cells were detected using CD3+ or CD19+ monoclonal antibody. (B) The percentages of CD4+ and CD8+ cells were detected using CD4+ or CD8+ monoclonal antibody.
41
Fig. 7. CAs induced transplanted H22 cells apoptosis in vivo. (A) Tumor cell cycle was analyzed by flow cytometry. (B) Effects of CAs on the histopathological examinations of
42
tumor tissues were observed by H&E staining (magnification, × 20). The white arrows in CAs groups represent apoptotic tumor cells nucleus bodies.
43
Fig. 8. Effects of CAs on Bcl-xL, Mcl-1, and activated caspase 3 expression in tumor tissues were measured by western blot, and the grayscale ratio of apoptosis-related proteins relative to β-actin protein was analyzed by Quantity One software. * p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
44
Table 1
The effects of CAs on body weight, number, and tumor inhibitory rate (TIR) of H22-bearing mice Group
Dose
Mice body weight (g)
Number of mice
TIR
(mg/kg)
Start
End
Start/end
(%)
Blank group
-
22.92 ± 1.01
31.91 ± 1.61**
10/ 10
Model group
-
22.22 ± 1.34
28.07 ± 1.38
10/ 9
CAs
50
22.81 ± 1.62
29.73 ± 1.37
10/ 10
29.28
CAs
250
22.00 ± 2.32
30.88 ± 1.65*
10/ 10
58.85
* p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
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Table 2
The percentages of tumor infiltrating CD3+ and CD8+ T lymphocytes in H22-bearing mice. Group
Dose (mg/kg)
CD3+ (%)
CD8+ (%)
Model group
-
77.24 ± 1.75
70.26 ± 4.79
CAs
50
83.67 ± 3.36*
76.09 ± 3.54
CAs
250
91.63 ± 3.47**
87.47 ± 2.48**
* p < 0.05 compared to model group was considered as significant. ** p < 0.01 compared to model group was considered as very significant.
46