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Anti-tumor and immunomodulatory activities induced by an alkali-extracted polysaccharide BCAP-1 from Bupleurum chinense via NF-κB signaling pathway
International Journal of Biological Macromolecules 95 (2017) 357–362
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Anti-tumor and immunomodulatory activities induced by an alkali-extracted polysaccharide BCAP-1 from Bupleurum chinense via NF-B signaling pathway Xiangfu Song a , Ting Ren b , Zhou Zheng b , Tiancheng Lu d , Zhicheng Wang a,∗ , Fengguo Du c,∗ , Haibin Tong b,∗ a
School of Public Health, Jilin University, Changchun 130021, China Jilin Provincial Key Laboratory of Molecular Geriatric Medicine, Life Science Research Center, Beihua University, Jilin 132013, China c College of Forestry, Beihua University, Jilin 132013, China d School of Life Science, Jilin Agricultural University, Changchun 130118, China b
a r t i c l e
i n f o
Article history: Received 19 August 2016 Received in revised form 30 September 2016 Accepted 17 October 2016 Available online 22 November 2016 Keywords: Polysaccharide Bupleurum chinense Immunomodulation
a b s t r a c t Bupleurum chinense is a well-known traditional Chinese medicine. Polysaccharides extracted from medical plants possess multiple healthy benefits. In the present study, an alkali-extracted polysaccharide (BCAP-1) was isolated from Bupleurum chinense, and evaluated its physicochemical features, anti-tumor activities and immunomodulatory effects. BCAP-1 was obtained by alkali-extraction, ethanol precipitation, and fractionation by DEAE-cellulose and Sepharose CL-6B columns. BCAP-1 markedly inhibited Sarcoma 180tumor growth in tumor-bearing mice, and increased the secretion of TNF-␣ in serum. MTT assay showed that BCAP-1 had no cytotoxicity against S-180 tumor cells. BCAP-1 enhanced the secretion of TNF-␣ and NO, and the transcripts of TNF-␣ and iNOS were increased. Meanwhile, BCAP-1 treatment induced the phosphorylation of p65 and decreased the expression of IB in macrophages. These results suggest that BCAP-1 could activate macrophages through NF-B signaling pathway, and the anti-tumor effects of BCAP-1 can be achieved by its immunostimulating features. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Bupleurum chinense is a well-known traditional Chinese medicine (TCM) that has been used for more than thousand years, and it distributes mainly in Hebei, Liaoning, Jilin, Heilongjiang and Neimeng province of China [1]. B. chinense was first mentioned in the Treatise on Cold Induced Febrile Disease (Shang Han Lun) as a primary ingredient of an ancient Chinese medicinal formula known as Xiao Chai Hu Tang from the 1st century BCE [2,3]. B. chinense is one of “harmony” herbs, balancing organs and energies within the body. It is also used as a tonic herb because of its ability to strengthen the action of the digestive tract, improve the function of liver and circulatory system, and relieve liver tension [4,5]. Active compounds isolated from B. chinense, such as saikosaponin, polysaccharide, flavonoid, essential oil and fatty acid, possess several pharmacological functions, including hepato-protective, mild
∗ Corresponding authors. E-mail addresses: [email protected] (Z. Wang), [email protected] (F. Du), [email protected] (H. Tong). http://dx.doi.org/10.1016/j.ijbiomac.2016.10.112 0141-8130/© 2016 Elsevier B.V. All rights reserved.
sedative, antipyretic, analgesic, anti-tussive, immunomodulatory effect, anti-fibrotic, anti-liver cancer and promoting liver regeneration [6–10]. In Japan, Korea and China, it has been widely prescribed to outpatients for treating chronic liver diseases. The enhancement or potentiation of host defense has been considered as a possible way of inhibiting tumor growth [11]. The majority of immunomodulatory compounds are polysaccharides, glycopeptide/protein complexes, and proteoglycans, and they can activate immunologic effector cells, including lymphocytes, macrophages, T cells, dendritic cells and natural killer cells involved in the innate and adaptive immunity, therefore, these immunomodulatory compounds are also called biologic response modifiers (BRMs) [12,13]. However, there is relatively little information pertaining to alkali-extracted polysaccharides from B. chinense, especially about the mechanism of immunomodulation and macrophage activation. Therefore, the purpose of the current study was to elucidate the underlying mechanisms of anti-tumor and macrophage activation of alkali-extracted polysaccharide isolated from the roots of B. chinense.
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2. Materials and methods 2.1. Materials and chemicals The roots of B. chinense were purchased from local medicine market in Jilin City (Jilin province, China), and identified according to the identification standard of Northeast Plant Retrieval List of China. Sepharose CL-6B gel and chemiluminescent (ECL) detection kit were purchased from Amersham Pharmacia Biotech. 5-fluorurazil (5-FU), T-series dextrans, bovine serum albumin (BSA), thioglycollate medium, Griess reagent, DEAE-cellulose, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lipopolysaccharide (LPS) were obtained from SigmaAldrich (St. Louis, MO, USA). RPMI 1640 medium was the product of Gibco (Grand Island, USA). Fetal calf serum (FCS) was provided by Hangzhou Sijiqing Corp. (Hangzhou, Zhejiang, China). Affi-Prep Polymyxin Matrix was purchased from BIO-RAD. All other chemical reagents were analytical grade. 2.2. Antibodies Anti-NF-B p65 antibody was obtained from Abcam. PhosphoNF-B p65 (Ser536) and IB-␣ antibodies were purchased from Cell Signaling Technology. -actin antibody was obtained from Santa Cruz Biotechnology. 2.3. Animals Male BALB/c mice (18–22 g) were purchased from Animal Experimental Center of Jilin University (Changchun, Jilin, China). The mice were housed in plastic cages at 22–24 ◦ C and 20% humidity with a 12 h light/dark cycle, and kept free access to tap water and food throughout the study. Animal experiments were conducted under the principles of laboratory animal care and approved by ethical committee for Laboratory Animals Care and Use of Jilin University. 2.4. Isolation and purification of alkali-extracted polysaccharides The roots of B. chinense were ground, and then extracted with 0.5 M NaOH solution. The alkali extract was filtered, centrifuged and neutralized with 0.1 M hydrochloric acid. The supernatant was concentrated and precipitated with 3-fold volumes of ethanol. Polysaccharide precipitate was collected by centrifugation and deproteinated by proteinase and Sevag method [14]. The supernatants were lyophilized to obtain crude alkaline B. chinense polysaccharides. The crude alkaline B. chinense polysaccharides were dissolved in distilled water, then loaded onto DEAE-cellulose column (3 × 30 cm), and eluted successively with distilled water and (0 → 1) M NaCl. The main fraction was collected, dialyzed, and lyophilized, and further fractioned on Sepharose CL-6B column (2.6 × 100 cm), eluted with 0.15 M NaCl to yield one main fraction, and coded as BCAP-1. Possible contaminants of endotoxin in BCAP-1 were removed using Affi-Prep Polymyxin Matrix (BIO-RAD).
Monosaccharide composition was identified and quantified using gas chromatography (GC). Briefly, BCAP-1 was hydrolyzed with 2 M trifluoroacetic acid (2 ml) at 120 ◦ C for 2 h. The hydrolyzed product was converted into the alditol acetates as described [18] and analyzed by GC. GC was performed on a Varian 3400 instrument (Hewlett-Packard Component, USA) equipped with DM-2330 capillary column (30 m × 0.32 mm × 0.2 m) and flame-ionization detector (FID). The homogeneity and molecular weight of BCAP-1 was evaluated by high performance gel permeation chromatography (HPGPC). BCAP-1 was dissolved in distilled water, and then applied to Shimadzu HPLC system equipped with a TSK-GEL G3000 PWXL column (7.8 × 300 mm), eluted with 0.1 M Na2 SO4 solution and detected by a RID-10A Refractive Index Detector (RID). The molecular weight of BCAP-1 was estimated by reference to the calibration curve. 2.6. In vivo antitumor activity and TNF-˛ secretion in serum Sixty mice were randomly divided into six groups. Sarcoma 180 cells (S-180) obtained from the peritoneal cavity of the tumorinoculated mice were washed and re-suspended in PBS. Then 0.2 ml of S-180 cell suspension (1 × 106 cells/ml) was inoculated into the right hind limbs at day 0. After 24 h, either BCAP-1 (100, 200, and 400 mg/kg, 0.2 ml), 5-fluorouracil (5-FU, 50 mg/kg) or sterile saline were orally administered daily. On the 21st day after tumor injection, all mice were weighed and sacrificed, and solid tumors, spleens and thymus were carefully extirpated and weighed. The tumor inhibition rate was calculated according to the following formula: %(inhibition ratio of tumor growth) = 100 − mean solid tumor weight of the treated group /mean solid tumor weight of the negative control group × 100
Relative thymus or spleen weight was measured in the ratio of the thymus or spleen weight (mg) to body weight (g). Tumor necrosis factor-alpha (TNF-␣) in serum collected from the tumor-bearing mice was measured using murine enzyme-linked immunosorbent assay (ELISA) kit. 2.7. MTT assay In vitro anti-tumor activity against S-180 cells was determined by MTT assay. Briefly, S-180 cells were seeded in 96-well plates for 24 h. Sterilized sample solutions were added into 96-well plates at final concentrations of 100, 200 and 400 g/ml for 72 h, while positive control group was treated with 5-FU (25 g/ml). 20 l MTT solution (5 mg/ml) was added in each well and incubated for 4 h, and then the supernatant was aspirated and 100 l DMSO was added. The absorbance was measured at 570 nm by Bio-Tek EXL800 microplate reader. 2.8. Preparation of peritoneal macrophages
2.5. General analytical methods Total carbohydrate content was determined by the phenolsulfuric acid colorimetric method [15]. Protein content was quantified according to the Bradford’s method [16]. Total uronic acid content was measured by the m-hydroxydiphenyl method [17]. Ultraviolet-visible spectra were recorded with a Varian Cary100 Spectrophotometer.
Male BALB/c mice were injected intraperitoneally with sterile thioglycollate medium for 3 consecutive days, and then the resident peritoneal macrophages were harvested by peritoneal lavage and centrifugation. Then peritoneal macrophages were incubated in cell culture dishes for 2 h. Non-adherent cells were removed by washing with PBS, the adhered macrophages were cultured for another 24 h with fresh complete RPMI-1640 medium.
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2.9. Measurement of phagocytosis capacity of peritoneal macrophage
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2.13. NF-B signaling activation Peritoneal macrophages were treated with BCAP-1 (100, 200 and 400 g/ml) in 6-well plates for 24 h. Cell pellets were lysed with RIPA lysis buffer and then centrifuged to remove cell debris. The supernatants were subjected to SDS-PAGE, and then proteins were transferred to NC membranes. The membranes were incubated with 5% nonfat milk in TBST, and then with primary antibodies (IB, p65 and phosphorylation of p65) and the HRP-conjugated secondary antibodies. Chemiluminescent detection was performed by using ECL plus western blotting reagents.
Peritoneal macrophages prepared as above were incubated with BCAP-1 (100, 200 and 400 g/ml) for 24 h, and then 100 l of aseptic neutral red solution were added (0.075%) into each well and incubated for another 1 h. After washed with PBS, the cells were lysed in 150 l cell lysis buffer (anhydrous ethanol:acetic acid = 1:1, v/v). The absorbance was measured at 550 nm using Bio-Tek EXL800 microplate reader. The absorbance represented the phagocytic ability of macrophages.
2.14. Statistical analysis
2.10. Assay of macrophage lysosomal phosphatase activity
All statistical analyses were performed with SPSS version 19.0 for Windows. Data were expressed as mean ± SEM and examined with one-way ANOVA. P-values of less than 0.05 were considered to be statistically significant.
Peritoneal macrophages in 96-well plates were treated with different concentrations of BCAP-1 (100, 200, and 400 g/ml) for 24 h, then solubilized with 25 l of 0.1% Triton X-100 and incubated for 30 min at room temperature. Then 100 l of 10 mM p-nitrophenyl phosphate was added to each well as substrate for acid phosphatase, followed by adding 0.1 M citrate buffer (50 l, pH 5.0). The cultures were further incubated for 30 min at 37 ◦ C, 0.2 M borate buffer (50 l, pH 9.8) was added to the mixture to terminate the reaction, and the absorbance at 405 nm was measured using microplate reader.
3. Results and discussion 3.1. Isolation, purification and physicochemical properties of BCAP-1 In the present study, the crude polysaccharide was obtained as a brown-coloured powder from B. chinense by hot alkali solution extraction. The yield of the crude polysaccharide was 13.2% of the dried material. After the freeze–thaw process and deproteinated by proteinase and Sevag method, the crude polysaccharide was purified by DEAE-cellulose ion exchange chromatography and Sepharose CL-6B gel filtration chromatography using ÄKTA explore 100 FPLC systems, the main fraction BCAP-1 was collected for further analysis. The total carbohydrate, protein, uronic acid contents, molecular weight and monosaccharide composition of BCAP-1 were summarized in Table 1. BCAP-1 had a negative response to Bradford assay and no absorption detected at 280 or 260 nm of UV spectrum, indicating the absence of protein and nucleic acid. Total carbohydrate content was 95.6% determined by the phenolsulfuric acid method, and BCAP-1 contained 15.3% uronic acid evaluated by the m-hydroxydiphenyl colorimetric method. The HPGPC profile of BCAP-1 showed a single and symmetrically sharp peak revealing that BCAP-1 was a homogeneous polysaccharide with an average molecular weight of 72.8 kDa. GC analysis showed BCAP-1 contained five types of monosaccharide, including arabinose, xylose, mannose, glucose and glucuronic acid in molar ratios of 1:3.2:0.7:3.6:1.2, indicating xylose and glucose was the predominant monosaccharides in BCAP-1.
2.11. Assay of TNF-˛ and NO secretion of peritoneal macrophages Peritoneal macrophages were incubated with BCAP-1 (100, 200 and 400 g/ml) in 96-well plates for 24 h, and then TNF-␣ secretion was measured using murine enzyme-linked immunosorbent assay (ELISA) kit. LPS (1 g/ml) was used as positive control. To measure NO, 100 l of cell-free culture medium was removed and placed into new 96-well plates. 100 l of Griess reagent was added into each well and incubated for 10 min at room temperature. The absorbance was measured at 540 nm using microplate reader. NO amount in each sample was calculated using a standard curve generated with sodium nitrite.
2.12. Semi-quantitative RT-PCR After incubated with BCAP-1 (100, 200 and 400 g/ml) in 6-well plates for 6 h, 12 h, and 24 h, the total RNA was isolated from the stimulated cells, and then reversely transcribed into cDNA using Promega RT-PCR kit. Primers used in the present study were as follows: TNF-␣ (forward 5 -TAGCCCACGTCGTAGCAAAC3 and reverse 5 -GCAGCCTTGTCCCTTGAAGA-3 ), iNOS (forward 5 -ACATCGACCCGTCCACATTAT-3 and reverse 5 -CAGAGGGGTAGGCTTGTCTC-3 ) and -actin (forward 5 ATGGAGGGGAATACAGCCC-3 and reverse 5 - TTCTTTGCAGCTC CTTCGTT-3 ). The cDNAs from the reverse transcription reaction were amplified under the following conditions: denaturation at 94 ◦ C for 30 s, annealing at 52 ◦ C for 30 s, and extension at 72 ◦ C for 30 s with a final extension at 72 ◦ C for 5 min. Amplified cDNA products were resolved on 1.5% agarose gel by electrophoresis and then stained with ethidium bromide.
3.2. In vivo tumor inhibitory activity of BCAP-1 As shown in Table 2, BCAP-1 showed significant inhibitory effect against S-180 solid tumor in vivo. The inhibitory rate reached 38.43% at the concentration of 400 mg/kg body weight. When treated with BCAP-1, a significant increase in spleen and thymus indices of S-180-bearing mice were also observed (Table 2). BCAP1 could also increase body weights of S-180-bearing mice in all BCAP-1-treated groups. Moreover, TNF-␣ is a principal mediator
Table 1 Physicochemical properties of BCAP-1. Sample
Mw (kDa)
Total sugar (%)
Protein (%)
Uronic acid (%)
BCAP-1
72.8
95.6
nda
15.3
a
nd: not detected.
Molar ratios of monosaccharide (mol%) Ara
Xyl
Man
Glc
GlcUA
1.0
3.2
0.7
3.6
1.2
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Table 2 In vivo anti-tumor activities of BCAP-1. Group Normal control Model control 5-FU BCAP-1
Dose (mg/kg)
100 200 400
Tumor weight (g) 2.42 ± 0.67 1.27 ± 0.31** 1.98 ± 0.63 1.71 ± 0.51* 1.49 ± 0.58**
Inhibition rate (%)
Relative spleen weight (mg/g)
Relative thymus weight (mg/g)
4.48 ± 0.31 3.93 ± 0.43 3.65 ± 0.71 4.21 ± 0.67 4.45 ± 0.45* 4.57 ± 0.68**
47.52 18.18 29.34 38.43
2.66 ± 0.24 2.42 ± 0.42 2.25 ± 0.41 2.53 ± 0.28 2.71 ± 0.36* 2.75 ± 0.45*
Values are expressed as mean ± SD (n = 10). * p < 0.05. ** p < 0.01 compared with control group.
Absorption at 550 nm
1.0 0.8 0.6
*
**
0.4 0.2 0.0
Control
LPS
100
200
400
μg/ml
BCAP-1
Fig. 1. TNF-␣ release in serum of S-180 tumor-bearing mice treated by BCAP-1. Values are mean ± SD; * P < 0.05, ** P < 0.01 vs. model control.
Fig. 3. Effects of BCAP-1 on phagocytosis activity of macrophage. Values are means ± SD; * p < 0.05, ** p < 0.01 vs. control.
in vitro, while the inhibition ratio of 5-FU reached 65.67% at the concentration of 50 g/ml. BCAP-1 effectively prevented the formation of S-180 tumor in vivo, consecutively, the level of TNF-␣ in serum was significantly increased in BCAP-1-treated mice. Unlike 5-FU, BCAP-1 had no significant direct cytotoxicity against S-180 cells. Much in vivo and in vitro evidence demonstrated that polysaccharides possess immunomodulating functions by stimulating both cellular and humoral immunoresponse [20–23]. Thymus and spleen are important immune organs, and thymus index and spleen index reflect the immune capability of the organism. In the present study, BCAP-1 promoted the weight of immune organs, spleen and thymus, of tumor-bearing mice. BCAP-1 could be an immunopotentiator to activate immune system. Therefore, in vivo anti-tumor activity of BCAP-1 may be due to activate mice’s own immune system to attack tumor cells. Fig. 2. Anti-tumor effect of BCAP-1 against S-180 in vitro. Values are mean ± SD; * P >0.05 vs. control.
3.3. Activation of peritoneal macrophage by BCAP-1
produced by macrophages involved in the regulation of necrosis, apoptosis and proliferation of multiple cell types. As shown in Fig. 1, the level of TNF-␣ in the serum of BCAP-1-treated S-180-bearing mice was elevated in a dose-dependent manner. 5-Fluorouracil (5-FU) is a chemotherapy medication, belongs to antimetabolite family, and widely used in the treatment of cancer [19]. 5-FU is a suicide inhibitor and works through irreversible inhibition of thymidylate synthase. In the present study, 5-FU exhibited a high in vivo tumor inhibitory rate (47.52%), but it decreased the relative spleen and thymus weights of S-180-bearing mice, as well as the level of TNF-␣ in serum. In addition, 5-FU also decreased the body weight of S-180-bearing mice, reflecting the toxicity to the body. We further evaluate whether BCAP-1 possessed a direct cytotoxicity against S-180 cells, then in vitro tumor inhibitory effects of BCAP-1 was determined by MTT assay. As shown in Fig. 2, BCAP1 exhibited only a weak cytotoxic activity against S-180 cells, and the highest inhibition ratio was 6.17% at the concentration of 400 g/ml, indicating that BCAP-1 had no obvious cytotoxicity
The immunologic action of BCAP-1 may begin with activating effector cells such as macrophages, lymphocytes, NK cells. The mononuclear phagocyte system (e.g., macrophages and monocytes) is indispensable for keeping homeostasis, and plays an essential role in host defense against tumor cells, presents antigens to lymphocytes, and release cytokines or mediators, including TNF-␣, interferons, interleukins, hydrolytic enzymes, bioactive lipids, and reactive oxygen species, destroy and inhibit tumor cell growth or activate other immunocytes [24,25]. Thus, we evaluated the effects of BCAP-1 on the activation of peritoneal macrophage. Phagocytic capacity is the most important function of activated macrophages. To assess the effects of BCAP-1 on phagocytosis, the phagocytic activity of macrophages to aseptic neutral red solution was examined in vitro. As shown in Fig. 3, BCAP-1 significantly promoted the phagocytic capacity of peritoneal macrophages. Compared with control group, phagocytic capacity was significantly increased by BCAP-1 treatment at the doses of 200 and 400 g/ml. Moreover, we also found that BCAP-1-stimulated macrophages had obvi-
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Table 3 Effects of BCAP-1 on immune function of peritoneal macrophages in vitro. Group Control LPS BCAP-1
Dose (g/ml)
Lysosomal enzyme activity (% of control)
100 200 400
100 271.4 ± 37.7** 131.7 ± 33.6 187.6 ± 52.1** 209.4 ± 51.7**
NO (M) 11.6 ± 3.4 37.2 ± 7.2** 17.4 ± 4.3 25.1 ± 6.4* 29.5 ± 5.6**
TNF-␣ (pg/ml) 131.61 ± 31.14 377.41 ± 57.19** 175.87 ± 42.42* 245.66 ± 49.24** 288.27 ± 61.31**
Values are expressed as mean ± SD. * p < 0.05. ** p < 0.01 compared with negative control group.
Fig. 4. Effect of BCAP-1 on the expression of TNF-␣ and iNOS at mRNAs level in peritoneal macrophages by RT-PCR. Representative images from three independent experiments are shown.
ously morphological changes from round shape to irregular shape. The above results revealed that BCAP-1 can induce macrophage activation by promoting phagocytic capacity. It is widely known that macrophages carry out their nonspecific defense functions of the elimination stage of the phagocytic process by activating the lysosomal phosphatase [26]. BCAP-1 could enhance lysosomal phosphatase activity (Table 3), the effects of BCAP-1 at the doses of 400 g/ml were comparable with those of LPS. The results suggest that BCAP-1 could activate lysosomal enzymes of macrophages. TNF-␣ is a principal mediator involved in the regulation of necrosis and apoptosis of tumor cells, and also induces the activation and chemotaxis of other leukocytes to attack tumors [27]. NO, synthesized from l-arginine by nitric oxide synthase (NOS), mediates a variety of biological functions as an intracellular signaling molecule, and NO production and iNOS activity through activated macrophages are considered the central roles in the regulation of immune response against tumors [28,29]. To examine whether the stimulation of BCAP-1 on peritoneal macrophages could influence the expression of cytokine, we measured TNF␣ and NO contents in the culture medium supernatants and the mRNA expression of TNF-␣ and iNOS. As shown in Table 3, BCAP1-treatment significantly increased the level of TNF-␣ in the culture medium in a dose-dependent manner, BCAP-1-activated peritoneal macrophages produced a large amount of TNF-␣ (288.27 pg/ml) at the concentration of 400 g/ml, over 2-fold than negative control. In addition, BCAP-1 treatment also resulted in a marked increase in NO production (29.5 M) at the concentration of 400 g/ml, almost 3 folds than negative control. Further, the gene expression of TNF-␣ and iNOS was detected at mRNA level by semi-quantitative RT-PCR. As shown in Fig. 4, the transcripts of TNF-␣ and iNOS were hardly detectable in unstimulated peritoneal macrophages. The amounts of TNF-␣ and iNOS transcript were increased significantly when exposure to BCAP-1 for 24 h. Macrophage activation is thought to be mediated primarily through the recognition between polysaccharides and pattern recognition receptors (PRRs), including Toll-like receptors, complement receptor 3 (CR3), dectin-1 and mannose receptor. Then initial signals trigger intracellular signaling cascades including tyrosine kinases, PKC, PI3K/Akt, MAPKs, and NF-B, eventually resulting in the transcriptional activation and gene expression of cytokines
Fig. 5. NF-B activation induced by BCAP-1. Peritoneal macrophages were exposed to BCAP-1 at 24 h. The whole cell lysates were probed with indicated antibodies. Representative images from three independent experiments are shown.
with anti-tumor activity [30,31]. NF-B family is an inducible and “rapid-acting” transcription factor stimulating cytokine gene expression, which plays an important role as critical regulator in host immune and inflammatory response after the recognition of polysaccharide by cell-surface receptors [32–34]. NF-B consists of heterodimeric protein complexes of which p65 and p50 subunits have been widely studied [35]. Inactive p65 is deposited with inhibitory IB proteins in the cytoplasm. When macrophages are activated, the upstream IKK kinases phosphorylate IB, leading to its ubiquitination and degradation, allowing p65 to translocate into the nucleus. At the same time, IKKs phosphorylate p65 on a critical serine (S536) modulating the transcriptional activity. We further investigated the phosphorylation of p65 protein (S536) and IB degradation in BCAP-1-stimulated macrophages using the Western blotting method. As shown in Fig. 5, small amount of p65 protein was phosphorylated in peritoneal macrophages of control group, whereas the p65 phosphorylation occurred after BCAP-1-treatment in a dose-dependent manner. The p65 phosphorylation level was markedly elevated at the concentration of 400 g/ml. In addition, there was a significant decrease of IB expression by BCAP-1 treatment than control group. Therefore, the increase of TNF-␣ and NO production induced by BCAP-1 might be related to the activation of NF-B signaling pathway. In conclusion, the present study demonstrated that BCAP-1, an alkali-extracted polysaccharide fraction from B. chinense, exhibited significant anti-tumor effects and immunostimulatory activities through NF-B signaling pathway. BCAP-1 can be considered as a new immunopotentiator to activate the immune system, and used as a protective therapy in cancer patients.
Acknowledgements This work was financially supported by National Natural Science Foundation of China (No. 31401203), Key Project of Science and Technology Department of Jilin Province (No. 20140204039YY), Youth Foundation of Health and Family Planning Commission of Jilin Province (2014Q040).
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Anti-tumor and immunomodulatory activities induced by an alkali-extracted polysaccharide BCAP-1 from Bupleurum chinense via NF-B signaling pathway Xiangfu Song a , Ting Ren b , Zhou Zheng b , Tiancheng Lu d , Zhicheng Wang a,∗ , Fengguo Du c,∗ , Haibin Tong b,∗ a
School of Public Health, Jilin University, Changchun 130021, China Jilin Provincial Key Laboratory of Molecular Geriatric Medicine, Life Science Research Center, Beihua University, Jilin 132013, China c College of Forestry, Beihua University, Jilin 132013, China d School of Life Science, Jilin Agricultural University, Changchun 130118, China b
a r t i c l e
i n f o
Article history: Received 19 August 2016 Received in revised form 30 September 2016 Accepted 17 October 2016 Available online 22 November 2016 Keywords: Polysaccharide Bupleurum chinense Immunomodulation
a b s t r a c t Bupleurum chinense is a well-known traditional Chinese medicine. Polysaccharides extracted from medical plants possess multiple healthy benefits. In the present study, an alkali-extracted polysaccharide (BCAP-1) was isolated from Bupleurum chinense, and evaluated its physicochemical features, anti-tumor activities and immunomodulatory effects. BCAP-1 was obtained by alkali-extraction, ethanol precipitation, and fractionation by DEAE-cellulose and Sepharose CL-6B columns. BCAP-1 markedly inhibited Sarcoma 180tumor growth in tumor-bearing mice, and increased the secretion of TNF-␣ in serum. MTT assay showed that BCAP-1 had no cytotoxicity against S-180 tumor cells. BCAP-1 enhanced the secretion of TNF-␣ and NO, and the transcripts of TNF-␣ and iNOS were increased. Meanwhile, BCAP-1 treatment induced the phosphorylation of p65 and decreased the expression of IB in macrophages. These results suggest that BCAP-1 could activate macrophages through NF-B signaling pathway, and the anti-tumor effects of BCAP-1 can be achieved by its immunostimulating features. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Bupleurum chinense is a well-known traditional Chinese medicine (TCM) that has been used for more than thousand years, and it distributes mainly in Hebei, Liaoning, Jilin, Heilongjiang and Neimeng province of China [1]. B. chinense was first mentioned in the Treatise on Cold Induced Febrile Disease (Shang Han Lun) as a primary ingredient of an ancient Chinese medicinal formula known as Xiao Chai Hu Tang from the 1st century BCE [2,3]. B. chinense is one of “harmony” herbs, balancing organs and energies within the body. It is also used as a tonic herb because of its ability to strengthen the action of the digestive tract, improve the function of liver and circulatory system, and relieve liver tension [4,5]. Active compounds isolated from B. chinense, such as saikosaponin, polysaccharide, flavonoid, essential oil and fatty acid, possess several pharmacological functions, including hepato-protective, mild
∗ Corresponding authors. E-mail addresses: [email protected] (Z. Wang), [email protected] (F. Du), [email protected] (H. Tong). http://dx.doi.org/10.1016/j.ijbiomac.2016.10.112 0141-8130/© 2016 Elsevier B.V. All rights reserved.
sedative, antipyretic, analgesic, anti-tussive, immunomodulatory effect, anti-fibrotic, anti-liver cancer and promoting liver regeneration [6–10]. In Japan, Korea and China, it has been widely prescribed to outpatients for treating chronic liver diseases. The enhancement or potentiation of host defense has been considered as a possible way of inhibiting tumor growth [11]. The majority of immunomodulatory compounds are polysaccharides, glycopeptide/protein complexes, and proteoglycans, and they can activate immunologic effector cells, including lymphocytes, macrophages, T cells, dendritic cells and natural killer cells involved in the innate and adaptive immunity, therefore, these immunomodulatory compounds are also called biologic response modifiers (BRMs) [12,13]. However, there is relatively little information pertaining to alkali-extracted polysaccharides from B. chinense, especially about the mechanism of immunomodulation and macrophage activation. Therefore, the purpose of the current study was to elucidate the underlying mechanisms of anti-tumor and macrophage activation of alkali-extracted polysaccharide isolated from the roots of B. chinense.
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2. Materials and methods 2.1. Materials and chemicals The roots of B. chinense were purchased from local medicine market in Jilin City (Jilin province, China), and identified according to the identification standard of Northeast Plant Retrieval List of China. Sepharose CL-6B gel and chemiluminescent (ECL) detection kit were purchased from Amersham Pharmacia Biotech. 5-fluorurazil (5-FU), T-series dextrans, bovine serum albumin (BSA), thioglycollate medium, Griess reagent, DEAE-cellulose, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lipopolysaccharide (LPS) were obtained from SigmaAldrich (St. Louis, MO, USA). RPMI 1640 medium was the product of Gibco (Grand Island, USA). Fetal calf serum (FCS) was provided by Hangzhou Sijiqing Corp. (Hangzhou, Zhejiang, China). Affi-Prep Polymyxin Matrix was purchased from BIO-RAD. All other chemical reagents were analytical grade. 2.2. Antibodies Anti-NF-B p65 antibody was obtained from Abcam. PhosphoNF-B p65 (Ser536) and IB-␣ antibodies were purchased from Cell Signaling Technology. -actin antibody was obtained from Santa Cruz Biotechnology. 2.3. Animals Male BALB/c mice (18–22 g) were purchased from Animal Experimental Center of Jilin University (Changchun, Jilin, China). The mice were housed in plastic cages at 22–24 ◦ C and 20% humidity with a 12 h light/dark cycle, and kept free access to tap water and food throughout the study. Animal experiments were conducted under the principles of laboratory animal care and approved by ethical committee for Laboratory Animals Care and Use of Jilin University. 2.4. Isolation and purification of alkali-extracted polysaccharides The roots of B. chinense were ground, and then extracted with 0.5 M NaOH solution. The alkali extract was filtered, centrifuged and neutralized with 0.1 M hydrochloric acid. The supernatant was concentrated and precipitated with 3-fold volumes of ethanol. Polysaccharide precipitate was collected by centrifugation and deproteinated by proteinase and Sevag method [14]. The supernatants were lyophilized to obtain crude alkaline B. chinense polysaccharides. The crude alkaline B. chinense polysaccharides were dissolved in distilled water, then loaded onto DEAE-cellulose column (3 × 30 cm), and eluted successively with distilled water and (0 → 1) M NaCl. The main fraction was collected, dialyzed, and lyophilized, and further fractioned on Sepharose CL-6B column (2.6 × 100 cm), eluted with 0.15 M NaCl to yield one main fraction, and coded as BCAP-1. Possible contaminants of endotoxin in BCAP-1 were removed using Affi-Prep Polymyxin Matrix (BIO-RAD).
Monosaccharide composition was identified and quantified using gas chromatography (GC). Briefly, BCAP-1 was hydrolyzed with 2 M trifluoroacetic acid (2 ml) at 120 ◦ C for 2 h. The hydrolyzed product was converted into the alditol acetates as described [18] and analyzed by GC. GC was performed on a Varian 3400 instrument (Hewlett-Packard Component, USA) equipped with DM-2330 capillary column (30 m × 0.32 mm × 0.2 m) and flame-ionization detector (FID). The homogeneity and molecular weight of BCAP-1 was evaluated by high performance gel permeation chromatography (HPGPC). BCAP-1 was dissolved in distilled water, and then applied to Shimadzu HPLC system equipped with a TSK-GEL G3000 PWXL column (7.8 × 300 mm), eluted with 0.1 M Na2 SO4 solution and detected by a RID-10A Refractive Index Detector (RID). The molecular weight of BCAP-1 was estimated by reference to the calibration curve. 2.6. In vivo antitumor activity and TNF-˛ secretion in serum Sixty mice were randomly divided into six groups. Sarcoma 180 cells (S-180) obtained from the peritoneal cavity of the tumorinoculated mice were washed and re-suspended in PBS. Then 0.2 ml of S-180 cell suspension (1 × 106 cells/ml) was inoculated into the right hind limbs at day 0. After 24 h, either BCAP-1 (100, 200, and 400 mg/kg, 0.2 ml), 5-fluorouracil (5-FU, 50 mg/kg) or sterile saline were orally administered daily. On the 21st day after tumor injection, all mice were weighed and sacrificed, and solid tumors, spleens and thymus were carefully extirpated and weighed. The tumor inhibition rate was calculated according to the following formula: %(inhibition ratio of tumor growth) = 100 − mean solid tumor weight of the treated group /mean solid tumor weight of the negative control group × 100
Relative thymus or spleen weight was measured in the ratio of the thymus or spleen weight (mg) to body weight (g). Tumor necrosis factor-alpha (TNF-␣) in serum collected from the tumor-bearing mice was measured using murine enzyme-linked immunosorbent assay (ELISA) kit. 2.7. MTT assay In vitro anti-tumor activity against S-180 cells was determined by MTT assay. Briefly, S-180 cells were seeded in 96-well plates for 24 h. Sterilized sample solutions were added into 96-well plates at final concentrations of 100, 200 and 400 g/ml for 72 h, while positive control group was treated with 5-FU (25 g/ml). 20 l MTT solution (5 mg/ml) was added in each well and incubated for 4 h, and then the supernatant was aspirated and 100 l DMSO was added. The absorbance was measured at 570 nm by Bio-Tek EXL800 microplate reader. 2.8. Preparation of peritoneal macrophages
2.5. General analytical methods Total carbohydrate content was determined by the phenolsulfuric acid colorimetric method [15]. Protein content was quantified according to the Bradford’s method [16]. Total uronic acid content was measured by the m-hydroxydiphenyl method [17]. Ultraviolet-visible spectra were recorded with a Varian Cary100 Spectrophotometer.
Male BALB/c mice were injected intraperitoneally with sterile thioglycollate medium for 3 consecutive days, and then the resident peritoneal macrophages were harvested by peritoneal lavage and centrifugation. Then peritoneal macrophages were incubated in cell culture dishes for 2 h. Non-adherent cells were removed by washing with PBS, the adhered macrophages were cultured for another 24 h with fresh complete RPMI-1640 medium.
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2.9. Measurement of phagocytosis capacity of peritoneal macrophage
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2.13. NF-B signaling activation Peritoneal macrophages were treated with BCAP-1 (100, 200 and 400 g/ml) in 6-well plates for 24 h. Cell pellets were lysed with RIPA lysis buffer and then centrifuged to remove cell debris. The supernatants were subjected to SDS-PAGE, and then proteins were transferred to NC membranes. The membranes were incubated with 5% nonfat milk in TBST, and then with primary antibodies (IB, p65 and phosphorylation of p65) and the HRP-conjugated secondary antibodies. Chemiluminescent detection was performed by using ECL plus western blotting reagents.
Peritoneal macrophages prepared as above were incubated with BCAP-1 (100, 200 and 400 g/ml) for 24 h, and then 100 l of aseptic neutral red solution were added (0.075%) into each well and incubated for another 1 h. After washed with PBS, the cells were lysed in 150 l cell lysis buffer (anhydrous ethanol:acetic acid = 1:1, v/v). The absorbance was measured at 550 nm using Bio-Tek EXL800 microplate reader. The absorbance represented the phagocytic ability of macrophages.
2.14. Statistical analysis
2.10. Assay of macrophage lysosomal phosphatase activity
All statistical analyses were performed with SPSS version 19.0 for Windows. Data were expressed as mean ± SEM and examined with one-way ANOVA. P-values of less than 0.05 were considered to be statistically significant.
Peritoneal macrophages in 96-well plates were treated with different concentrations of BCAP-1 (100, 200, and 400 g/ml) for 24 h, then solubilized with 25 l of 0.1% Triton X-100 and incubated for 30 min at room temperature. Then 100 l of 10 mM p-nitrophenyl phosphate was added to each well as substrate for acid phosphatase, followed by adding 0.1 M citrate buffer (50 l, pH 5.0). The cultures were further incubated for 30 min at 37 ◦ C, 0.2 M borate buffer (50 l, pH 9.8) was added to the mixture to terminate the reaction, and the absorbance at 405 nm was measured using microplate reader.
3. Results and discussion 3.1. Isolation, purification and physicochemical properties of BCAP-1 In the present study, the crude polysaccharide was obtained as a brown-coloured powder from B. chinense by hot alkali solution extraction. The yield of the crude polysaccharide was 13.2% of the dried material. After the freeze–thaw process and deproteinated by proteinase and Sevag method, the crude polysaccharide was purified by DEAE-cellulose ion exchange chromatography and Sepharose CL-6B gel filtration chromatography using ÄKTA explore 100 FPLC systems, the main fraction BCAP-1 was collected for further analysis. The total carbohydrate, protein, uronic acid contents, molecular weight and monosaccharide composition of BCAP-1 were summarized in Table 1. BCAP-1 had a negative response to Bradford assay and no absorption detected at 280 or 260 nm of UV spectrum, indicating the absence of protein and nucleic acid. Total carbohydrate content was 95.6% determined by the phenolsulfuric acid method, and BCAP-1 contained 15.3% uronic acid evaluated by the m-hydroxydiphenyl colorimetric method. The HPGPC profile of BCAP-1 showed a single and symmetrically sharp peak revealing that BCAP-1 was a homogeneous polysaccharide with an average molecular weight of 72.8 kDa. GC analysis showed BCAP-1 contained five types of monosaccharide, including arabinose, xylose, mannose, glucose and glucuronic acid in molar ratios of 1:3.2:0.7:3.6:1.2, indicating xylose and glucose was the predominant monosaccharides in BCAP-1.
2.11. Assay of TNF-˛ and NO secretion of peritoneal macrophages Peritoneal macrophages were incubated with BCAP-1 (100, 200 and 400 g/ml) in 96-well plates for 24 h, and then TNF-␣ secretion was measured using murine enzyme-linked immunosorbent assay (ELISA) kit. LPS (1 g/ml) was used as positive control. To measure NO, 100 l of cell-free culture medium was removed and placed into new 96-well plates. 100 l of Griess reagent was added into each well and incubated for 10 min at room temperature. The absorbance was measured at 540 nm using microplate reader. NO amount in each sample was calculated using a standard curve generated with sodium nitrite.
2.12. Semi-quantitative RT-PCR After incubated with BCAP-1 (100, 200 and 400 g/ml) in 6-well plates for 6 h, 12 h, and 24 h, the total RNA was isolated from the stimulated cells, and then reversely transcribed into cDNA using Promega RT-PCR kit. Primers used in the present study were as follows: TNF-␣ (forward 5 -TAGCCCACGTCGTAGCAAAC3 and reverse 5 -GCAGCCTTGTCCCTTGAAGA-3 ), iNOS (forward 5 -ACATCGACCCGTCCACATTAT-3 and reverse 5 -CAGAGGGGTAGGCTTGTCTC-3 ) and -actin (forward 5 ATGGAGGGGAATACAGCCC-3 and reverse 5 - TTCTTTGCAGCTC CTTCGTT-3 ). The cDNAs from the reverse transcription reaction were amplified under the following conditions: denaturation at 94 ◦ C for 30 s, annealing at 52 ◦ C for 30 s, and extension at 72 ◦ C for 30 s with a final extension at 72 ◦ C for 5 min. Amplified cDNA products were resolved on 1.5% agarose gel by electrophoresis and then stained with ethidium bromide.
3.2. In vivo tumor inhibitory activity of BCAP-1 As shown in Table 2, BCAP-1 showed significant inhibitory effect against S-180 solid tumor in vivo. The inhibitory rate reached 38.43% at the concentration of 400 mg/kg body weight. When treated with BCAP-1, a significant increase in spleen and thymus indices of S-180-bearing mice were also observed (Table 2). BCAP1 could also increase body weights of S-180-bearing mice in all BCAP-1-treated groups. Moreover, TNF-␣ is a principal mediator
Table 1 Physicochemical properties of BCAP-1. Sample
Mw (kDa)
Total sugar (%)
Protein (%)
Uronic acid (%)
BCAP-1
72.8
95.6
nda
15.3
a
nd: not detected.
Molar ratios of monosaccharide (mol%) Ara
Xyl
Man
Glc
GlcUA
1.0
3.2
0.7
3.6
1.2
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Table 2 In vivo anti-tumor activities of BCAP-1. Group Normal control Model control 5-FU BCAP-1
Dose (mg/kg)
100 200 400
Tumor weight (g) 2.42 ± 0.67 1.27 ± 0.31** 1.98 ± 0.63 1.71 ± 0.51* 1.49 ± 0.58**
Inhibition rate (%)
Relative spleen weight (mg/g)
Relative thymus weight (mg/g)
4.48 ± 0.31 3.93 ± 0.43 3.65 ± 0.71 4.21 ± 0.67 4.45 ± 0.45* 4.57 ± 0.68**
47.52 18.18 29.34 38.43
2.66 ± 0.24 2.42 ± 0.42 2.25 ± 0.41 2.53 ± 0.28 2.71 ± 0.36* 2.75 ± 0.45*
Values are expressed as mean ± SD (n = 10). * p < 0.05. ** p < 0.01 compared with control group.
Absorption at 550 nm
1.0 0.8 0.6
*
**
0.4 0.2 0.0
Control
LPS
100
200
400
μg/ml
BCAP-1
Fig. 1. TNF-␣ release in serum of S-180 tumor-bearing mice treated by BCAP-1. Values are mean ± SD; * P < 0.05, ** P < 0.01 vs. model control.
Fig. 3. Effects of BCAP-1 on phagocytosis activity of macrophage. Values are means ± SD; * p < 0.05, ** p < 0.01 vs. control.
in vitro, while the inhibition ratio of 5-FU reached 65.67% at the concentration of 50 g/ml. BCAP-1 effectively prevented the formation of S-180 tumor in vivo, consecutively, the level of TNF-␣ in serum was significantly increased in BCAP-1-treated mice. Unlike 5-FU, BCAP-1 had no significant direct cytotoxicity against S-180 cells. Much in vivo and in vitro evidence demonstrated that polysaccharides possess immunomodulating functions by stimulating both cellular and humoral immunoresponse [20–23]. Thymus and spleen are important immune organs, and thymus index and spleen index reflect the immune capability of the organism. In the present study, BCAP-1 promoted the weight of immune organs, spleen and thymus, of tumor-bearing mice. BCAP-1 could be an immunopotentiator to activate immune system. Therefore, in vivo anti-tumor activity of BCAP-1 may be due to activate mice’s own immune system to attack tumor cells. Fig. 2. Anti-tumor effect of BCAP-1 against S-180 in vitro. Values are mean ± SD; * P >0.05 vs. control.
3.3. Activation of peritoneal macrophage by BCAP-1
produced by macrophages involved in the regulation of necrosis, apoptosis and proliferation of multiple cell types. As shown in Fig. 1, the level of TNF-␣ in the serum of BCAP-1-treated S-180-bearing mice was elevated in a dose-dependent manner. 5-Fluorouracil (5-FU) is a chemotherapy medication, belongs to antimetabolite family, and widely used in the treatment of cancer [19]. 5-FU is a suicide inhibitor and works through irreversible inhibition of thymidylate synthase. In the present study, 5-FU exhibited a high in vivo tumor inhibitory rate (47.52%), but it decreased the relative spleen and thymus weights of S-180-bearing mice, as well as the level of TNF-␣ in serum. In addition, 5-FU also decreased the body weight of S-180-bearing mice, reflecting the toxicity to the body. We further evaluate whether BCAP-1 possessed a direct cytotoxicity against S-180 cells, then in vitro tumor inhibitory effects of BCAP-1 was determined by MTT assay. As shown in Fig. 2, BCAP1 exhibited only a weak cytotoxic activity against S-180 cells, and the highest inhibition ratio was 6.17% at the concentration of 400 g/ml, indicating that BCAP-1 had no obvious cytotoxicity
The immunologic action of BCAP-1 may begin with activating effector cells such as macrophages, lymphocytes, NK cells. The mononuclear phagocyte system (e.g., macrophages and monocytes) is indispensable for keeping homeostasis, and plays an essential role in host defense against tumor cells, presents antigens to lymphocytes, and release cytokines or mediators, including TNF-␣, interferons, interleukins, hydrolytic enzymes, bioactive lipids, and reactive oxygen species, destroy and inhibit tumor cell growth or activate other immunocytes [24,25]. Thus, we evaluated the effects of BCAP-1 on the activation of peritoneal macrophage. Phagocytic capacity is the most important function of activated macrophages. To assess the effects of BCAP-1 on phagocytosis, the phagocytic activity of macrophages to aseptic neutral red solution was examined in vitro. As shown in Fig. 3, BCAP-1 significantly promoted the phagocytic capacity of peritoneal macrophages. Compared with control group, phagocytic capacity was significantly increased by BCAP-1 treatment at the doses of 200 and 400 g/ml. Moreover, we also found that BCAP-1-stimulated macrophages had obvi-
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Table 3 Effects of BCAP-1 on immune function of peritoneal macrophages in vitro. Group Control LPS BCAP-1
Dose (g/ml)
Lysosomal enzyme activity (% of control)
100 200 400
100 271.4 ± 37.7** 131.7 ± 33.6 187.6 ± 52.1** 209.4 ± 51.7**
NO (M) 11.6 ± 3.4 37.2 ± 7.2** 17.4 ± 4.3 25.1 ± 6.4* 29.5 ± 5.6**
TNF-␣ (pg/ml) 131.61 ± 31.14 377.41 ± 57.19** 175.87 ± 42.42* 245.66 ± 49.24** 288.27 ± 61.31**
Values are expressed as mean ± SD. * p < 0.05. ** p < 0.01 compared with negative control group.
Fig. 4. Effect of BCAP-1 on the expression of TNF-␣ and iNOS at mRNAs level in peritoneal macrophages by RT-PCR. Representative images from three independent experiments are shown.
ously morphological changes from round shape to irregular shape. The above results revealed that BCAP-1 can induce macrophage activation by promoting phagocytic capacity. It is widely known that macrophages carry out their nonspecific defense functions of the elimination stage of the phagocytic process by activating the lysosomal phosphatase [26]. BCAP-1 could enhance lysosomal phosphatase activity (Table 3), the effects of BCAP-1 at the doses of 400 g/ml were comparable with those of LPS. The results suggest that BCAP-1 could activate lysosomal enzymes of macrophages. TNF-␣ is a principal mediator involved in the regulation of necrosis and apoptosis of tumor cells, and also induces the activation and chemotaxis of other leukocytes to attack tumors [27]. NO, synthesized from l-arginine by nitric oxide synthase (NOS), mediates a variety of biological functions as an intracellular signaling molecule, and NO production and iNOS activity through activated macrophages are considered the central roles in the regulation of immune response against tumors [28,29]. To examine whether the stimulation of BCAP-1 on peritoneal macrophages could influence the expression of cytokine, we measured TNF␣ and NO contents in the culture medium supernatants and the mRNA expression of TNF-␣ and iNOS. As shown in Table 3, BCAP1-treatment significantly increased the level of TNF-␣ in the culture medium in a dose-dependent manner, BCAP-1-activated peritoneal macrophages produced a large amount of TNF-␣ (288.27 pg/ml) at the concentration of 400 g/ml, over 2-fold than negative control. In addition, BCAP-1 treatment also resulted in a marked increase in NO production (29.5 M) at the concentration of 400 g/ml, almost 3 folds than negative control. Further, the gene expression of TNF-␣ and iNOS was detected at mRNA level by semi-quantitative RT-PCR. As shown in Fig. 4, the transcripts of TNF-␣ and iNOS were hardly detectable in unstimulated peritoneal macrophages. The amounts of TNF-␣ and iNOS transcript were increased significantly when exposure to BCAP-1 for 24 h. Macrophage activation is thought to be mediated primarily through the recognition between polysaccharides and pattern recognition receptors (PRRs), including Toll-like receptors, complement receptor 3 (CR3), dectin-1 and mannose receptor. Then initial signals trigger intracellular signaling cascades including tyrosine kinases, PKC, PI3K/Akt, MAPKs, and NF-B, eventually resulting in the transcriptional activation and gene expression of cytokines
Fig. 5. NF-B activation induced by BCAP-1. Peritoneal macrophages were exposed to BCAP-1 at 24 h. The whole cell lysates were probed with indicated antibodies. Representative images from three independent experiments are shown.
with anti-tumor activity [30,31]. NF-B family is an inducible and “rapid-acting” transcription factor stimulating cytokine gene expression, which plays an important role as critical regulator in host immune and inflammatory response after the recognition of polysaccharide by cell-surface receptors [32–34]. NF-B consists of heterodimeric protein complexes of which p65 and p50 subunits have been widely studied [35]. Inactive p65 is deposited with inhibitory IB proteins in the cytoplasm. When macrophages are activated, the upstream IKK kinases phosphorylate IB, leading to its ubiquitination and degradation, allowing p65 to translocate into the nucleus. At the same time, IKKs phosphorylate p65 on a critical serine (S536) modulating the transcriptional activity. We further investigated the phosphorylation of p65 protein (S536) and IB degradation in BCAP-1-stimulated macrophages using the Western blotting method. As shown in Fig. 5, small amount of p65 protein was phosphorylated in peritoneal macrophages of control group, whereas the p65 phosphorylation occurred after BCAP-1-treatment in a dose-dependent manner. The p65 phosphorylation level was markedly elevated at the concentration of 400 g/ml. In addition, there was a significant decrease of IB expression by BCAP-1 treatment than control group. Therefore, the increase of TNF-␣ and NO production induced by BCAP-1 might be related to the activation of NF-B signaling pathway. In conclusion, the present study demonstrated that BCAP-1, an alkali-extracted polysaccharide fraction from B. chinense, exhibited significant anti-tumor effects and immunostimulatory activities through NF-B signaling pathway. BCAP-1 can be considered as a new immunopotentiator to activate the immune system, and used as a protective therapy in cancer patients.
Acknowledgements This work was financially supported by National Natural Science Foundation of China (No. 31401203), Key Project of Science and Technology Department of Jilin Province (No. 20140204039YY), Youth Foundation of Health and Family Planning Commission of Jilin Province (2014Q040).
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